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  • 1. Ahlqvist, J.
    et al.
    Dainiak, M.B.
    Kumar, A.
    Hornsten, Gunnar E.
    Galaev, I.Yu.
    Mattiasson, B.
    Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 354, no 2, p. 229-237Article in journal (Refereed)
    Abstract [en]

    A novel minicolumn chromatographic method to monitor the production of inclusion bodies during fermentation and an enzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced as inclusion bodies was labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed us to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed on the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary IgG and of enzymatic reaction within the adsorbent. © 2006 Elsevier Inc. All rights reserved.

  • 2.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    Gustafsson, A. C.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Single-nucleotide polymorphism analysis by pyrosequencing2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 280, no 1, p. 103-110Article in journal (Refereed)
    Abstract [en]

    There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic: alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

  • 3.
    Andersson, Henrik
    et al.
    Attana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden/The Ångström Laboratory, Solid State Electronics, Uppsala University, P.O. Box 534, SE-751 21 Uppsala, Sweden.
    Myrskog, Annika
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Ingemarsson, Björn
    Attana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden.
    Pei, Zhichao
    Attana AB, Björnnäsvägen 21, SE-114 19 Stockholm, Sweden.
    Optimizing immobilization conditions on a two dimensional carboxylbiosensor surface: pH dependence of antibody orientation andantigen binding capacity2009In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309Article in journal (Other academic)
    Abstract [en]

    The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We have shown that successful immobilization is highly dependent on surface pKa, antibody pI and pH of immobilization buffer. By use of EDC/sulfo-NHS activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization also at very low pH has been made possible which is of importance for immobilization of acidic proteins. Generic immobilization conditions were demonstrated on a panel of antibodies which resulted in an average coefficient of variation of 4% for the immobilization of these antibodies.

    Antigen binding capacity as a function of immobilization pH was studied. In most cases the antigen binding capacity followed the immobilization response. However, the antigen to antibody binding ratio differed between the antibodies investigated, and for one of the antibodies, the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab antibodies on different antibody surfaces showed that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

  • 4. Andersson, R. M.
    et al.
    Carlsson, Kjell
    KTH, Superseded Departments, Physics.
    Liljeborg, A.
    Brismar, Hjalmar
    Characterization of probe binding and comparison of its influence on fluorescence lifetime of two pH-sensitive benzo c xanthene dyes using intensity-modulated multiple-wavelength scanning technique2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 283, no 1, p. 104-110Article in journal (Refereed)
    Abstract [en]

    Quantitative pH imaging using the carboxy semi-naphthofluorescein dyes SNAFL-1 and SNAFL-2 can be performed by measurement of intensity ratios or fluorescence lifetimes. However, there is a controversy as to whether the latter method has the practical advantage of a straightforward pH calibration in buffers compared to a cumbersome and time-consuming procedure in cells. In this study we have undertaken a systematic study of the potential factors influencing the fluorescence lifetime of the probes at different pH using confocal microscopy. In vitro results demonstrate that factors such as lipid and protein concentrations have a substantial influence on pH measurements based on fluorescence lifetime. The pH could be overestimated by more than 2 pH units. Studies in permeabilized COS-7 cells demonstrate the same trends as observed in the in vitro studies.

  • 5.
    Banerjee, S
    et al.
    Department of Polymer Science and Technology, University of Calcutta, India.
    Sarkar, Priya
    Department of Polymer Science and Technology, University of Calcutta, India.
    Turner, Anthony
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, The Institute of Technology.
    Amperometric Biosensor for estimation of Glucose-6-phosphate Using Prussian Blue Nanoparticles.2013In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 439, no 2, p. 194-200Article in journal (Refereed)
    Abstract [en]

    Glucose-6-phosphateplays an important role in carbohydrate metabolism of all living organisms.Compared to the conventional analytical methods available for estimation of glucose-6-phosphate,the biosensors having relative simplicity, specificity, low-cost and fastresponse time are a promising alternative. We have reported a glucose-6-phosphatesensor based on screen-printed electrode utilizing Prussian blue nanoparticlesand enzymes, glucose-6-phosphate dehydrogenase and glutathione reductase. The Prussianblue nanoparticles acted as a mediator enhancing the rate of electrochemical responses.The Fourier transforminfrared spectroscopy and energy-dispersiveX-ray spectroscopy study confirmed the formation of Prussian blue, whereas, the atomic forced microscopy revealed that Prussian bluenanoparticles were about 25-30 nm in diameter. To obtainmaximum amperometric response, optimization studies were conducted for pH,enzyme and cofactor loading. The proposed glucose-6-phosphate biosensor showed goodstability, rapid response time and broad linear response in the range of 0.01-1.25mM and detection limit of 6.3 mM. The biosensor also worked well for serum samples and exhibitedexcellent anti-interference ability.

  • 6.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems: Diffusion through an inhibitor barrier2018In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 546, p. 43-49Article in journal (Refereed)
    Abstract [en]

    A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

  • 7.
    Boija, Elisabet
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Lundquist, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Evaluation of bilayer disks as plant cell membrane models in partition studies2007In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 364, no 2, p. 145-152Article in journal (Refereed)
    Abstract [en]

    We have studied the partitioning of a set of phenolic compounds used as lignin precursor models into lipid bilayer disks and liposomes. The bilayer disks are open bilayer structures stabilized by polyethylene glycol-conjugated lipids. Our results indicate that disks generate more accurate partition data than do liposomes. Furthermore, we show that the partitioning into the membrane phase is reduced slightly if disks composed of 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol are exchanged for disks with a lipid composition mimicking that of the root tissue of Zea mays L.

  • 8.
    Bondeson, K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Frostell-Karlsson, A
    Fägerstam, L
    Magnusson, G
    Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor.1993In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 214, no 1, p. 245-51Article in journal (Refereed)
    Abstract [en]

    Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.

  • 9.
    Bruce, Stephen J
    et al.
    Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Jonsson, Pär
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Antti, Henrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cloarec, Olivier
    Technologie Servier, 45000 Orleans, France.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Marklund, Stefan L
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Moritz, Thomas
    Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis2009In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 372, no 2, p. 237-249Article in journal (Refereed)
    Abstract [en]

    The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS–DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.

  • 10. Christopeit, Tony
    et al.
    Gossas, Thomas
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor2009In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 391, no 1, p. 39-44Article in journal (Refereed)
    Abstract [en]

    The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K-D = 0.03 mM and a low-affinity site with K-D = 5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K-D = 5 mu M at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and Suggest that Ca2+ is an efficient regulator under physiological conditions.

  • 11.
    Christopeit, Tony
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nyström, Susanne
    Baraznenok, Vera
    Lindström, Erik
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 414, no 1, p. 14-22Article in journal (Refereed)
    Abstract [en]

    A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.

  • 12.
    Cilpa-Karhu, Geraldine
    et al.
    Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki.
    Lipponen, Katriina
    Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki.
    Samuelsson, Jörgen
    Karlstad University, Faculty of Health, Science and Technology (starting 2013), Department of Engineering and Chemical Sciences.
    Ööri, Katariina
    Wihuri Research Institute, FIN-00290 Helsinki, Finland.
    Fornstedt, Torgny
    Karlstad University, Faculty of Health, Science and Technology (starting 2013), Department of Engineering and Chemical Sciences.
    Riekkola, Marja-Liisa
    Chemistry, Department of Chemistry, University of Helsinki.
    Three complementary techniques for the clarification of temperature effect on low-density lipoprotein–chondroitin-6-sulfate interaction2013In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 443, no 2, p. 139-147Article in journal (Refereed)
    Abstract [en]

    Abstract A rigorous processing of adsorption data from quartz crystal microbalance technology was successfully combined with the data obtained by partial filling affinity capillary electrophoresis and molecular dynamics for the clarification of the temperature effect on the interaction of a major glycosaminoglycan chain chondroitin-6-sulfate (C6S) of proteoglycans with low-density lipoprotein (LDL) and with a peptide fragment of apolipoprotein B-100 (residues 3359–3377 of LDL, PPBS). Two experimental techniques and computational atomistic methods demonstrated a nonlinear pattern of the affinity of C6S at temperatures above 38.0 °C to both LDL and PPBS. The temperature affects the interaction of C6S with LDL and PPBS by influencing the structural behavior of glycosaminoglycan C6S and/or that of LDL.

  • 13.
    Duong-Thi, Minh-Dao
    et al.
    Linnaeus Univ, Dept Chem & Biomed Sci, SE-39182 Kalmar, Sweden.
    Bergström, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, The Institute of Technology.
    Mandenius, Carl-Fredrik
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, The Institute of Technology.
    Bergstrom, Maria
    Linnaeus University, Sweden .
    Fex, Tomas
    University of Gothenburg, Sweden .
    Ohlson, Sten
    Nanyang Technology University, Singapore .
    Comparison of weak affinity chromatography and surface plasmon resonance in determining affinity of small molecules2014In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 461, p. 57-59Article in journal (Refereed)
    Abstract [en]

    In this study, we compared affinity data from surface plasmon resonance (SPR) and weak affinity chromatography (WAC), two established techniques for determination of weak affinity (mM-mu M) small molecule-protein interactions. In the current comparison, thrombin was used as target protein. In WAC the affinity constant (K-D) was determined from retention times, and in SPR it was determined by Langmuir isotherm fitting of steady-state responses. Results indicate a strong correlation between the two methods (R-2 = 0.995, P less than 0.0001).

  • 14.
    Duong-Thi, Minh-Dao
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Bergström, Gunnar
    Linköping Univ, Sweden.
    Mandenius, Carl-Fredrik
    Linköping Univ, Sweden.
    Bergström, Maria
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Fex, Tomas
    Univ Gothenburg, Sweden.
    Ohlson, Sten
    Nanyang Technol Univ, Singapore.
    Comparison of weak affinity chromatography and surface plasmon resonance in determining affinity of small molecules2014In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 461, p. 57-59Article in journal (Refereed)
    Abstract [en]

    In this study, we compared affinity data from surface plasmon resonance (SPR) and weak affinity chromatography (WAC), two established techniques for determination of weak affinity (mM-mu M) small molecule-protein interactions. In the current comparison, thrombin was used as target protein. In WAC the affinity constant (K-D) was determined from retention times, and in SPR it was determined by Langmuir isotherm fitting of steady-state responses. Results indicate a strong correlation between the two methods (R-2 = 0.995, P < 0.0001). (C) 2014 Elsevier Inc. All rights reserved.

  • 15.
    Duong-Thi, Minh-Dao
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Meiby, Elinor
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Bergström, Maria
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Fex, Tomas
    Isaksson, Roland
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Ohlson, Sten
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Weak affinity chromatography as a new approach for fragment screening in drug discovery2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 414, no 1, p. 138-146Article in journal (Refereed)
    Abstract [en]

    Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–uM in dissociation constant (KD)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23 compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10uM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.

  • 16.
    Eberle, Andrea B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Böhm, Stefanie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Farrants, Ann-Kristin Östlund
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation2012In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 426, no 2, p. 147-152Article in journal (Refereed)
    Abstract [en]

    Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate the interactions between proteins and DNA in vivo. ChIP is often used as a quantitative tool, and proper quantification relies on the use of adequate references for data normalization. However, many ChIP experiments involve analyses of samples that have been submitted to experimental treatments with unknown effects, and this precludes the choice of suitable internal references. We have developed a normalization method based on the use of a synthetic DNA-antibody complex that can be used as an external reference instead. A fixed amount of this synthetic DNA-antibody complex is spiked into the chromatin extract at the beginning of the ChIP experiment. The DNA-antibody complex is isolated together with the sample of interest, and the amounts of synthetic DNA recovered in each tube are measured at the end of the process. The yield of synthetic DNA recovery in each sample is then used to normalize the results obtained with the antibodies of interest. Using this approach, we could compensate for losses of material, reduce the variability between ChIP replicates, and increase the accuracy and statistical resolution of the data.

  • 17.
    Ehn, Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nourizad, Nader
    KTH, Superseded Departments, Biotechnology.
    Bergstrom, Kristina
    KTH, Superseded Departments, Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 329, no 1, p. 11-20Article in journal (Refereed)
    Abstract [en]

    Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.

  • 18.
    Ekman, Pia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Jäger, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Quantification of subnanomolar amounts of phosphate bound to seryl and threonyl residues in phosphoproteins using alkaline hydrolysis and malachite green1993In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 214, no 1, p. 138-141Article in journal (Refereed)
    Abstract [en]

    An assay for protein-bound phosphate with a capacity to determine 100 pmol of phosphate is described. It is based on the combination of two well known methods: the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoproteins and the quantification of the released phosphate by the use of malachite green and phosphomolybdate.

  • 19. Eriksson, J.
    et al.
    Karamohamed, S.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Method for real-time detection of inorganic pyrophosphatase activity2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 293, no 1, p. 67-70Article in journal (Refereed)
    Abstract [en]

    A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.

  • 20.
    Eriksson, Jonas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Biotechnology, Royal Institute of Technology, Stockholm.
    Karamohamed, S
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts.
    Nyrén, P
    Department of Biotechnology, Royal Institute of Technology, Stockholm.
    Method for real-time detection of inorganic pyrophosphatase activity2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 293, no 1, p. 67-70Article in journal (Refereed)
    Abstract [en]

    A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.

  • 21.
    Eriksson, Jonas
    et al.
    Department of Biotechnology, AlbaNova University Center, SCFAB, Royal Institute of Technology, Stockholm.
    Nordström, Tommy
    Department of Biotechnology, AlbaNova University Center, SCFAB, Royal Institute of Technology, Stockholm.
    Nyrén, Pål
    Department of Biotechnology, AlbaNova University Center, SCFAB, Royal Institute of Technology, Stockholm.
    Method enabling firefly luciferase-based bioluminometric assays at elevated temperatures2003In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 314, no 1, p. 158-161Article in journal (Refereed)
  • 22. Fedulova, Natalia
    et al.
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Experimental conditions affecting functional comparison of highly active glutathione transferases2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, no 1, p. 16-23Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. The combination of time-dependent and enzyme concentration-dependent loss of activity and the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

  • 23.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, no 1, p. 16-23Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. Combination of time-dependent and enzyme concentration-dependent loss of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurementsof GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

  • 24. Frykholm, Karolin
    et al.
    Freitag, Camilla
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Tegenfeldt, Jonas O.
    Graneli, Annette
    Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad512013In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 443, no 2, p. 261-268Article in journal (Refereed)
    Abstract [en]

    Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.

  • 25.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Liu, Yanling
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gene-based identification of bacterial colonies with an electric chip2005In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, no 2, p. 270-276Article in journal (Refereed)
    Abstract [en]

    A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.

  • 26.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, Superseded Departments, Biotechnology.
    Los, M.
    Holmgren, Anders
    KTH, Superseded Departments, Fibre and Polymer Technology.
    Albers, J.
    Czyz, A.
    Hintsche, R.
    Wegrzyn, G.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 324, no 1, p. 84-91Article in journal (Refereed)
    Abstract [en]

    Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample.. These analyses confirmed the specificity of the assay.

  • 27.
    Garscha, Ulrike
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Oliw, Ernst H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases2007In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 367, no 2, p. 238-246Article in journal (Refereed)
    Abstract [en]

    8-Hydroxyoctadeca-9Z,12Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8E,12Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10S-HODE by Lentinula edodes, and 10R-HODE by Epichloe typhina. Racemic [8-2H]8-HODE and [10-2H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess–Martin periodinane followed by reduction to hydroxy fatty acids with NaB2H4. The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) with 8R-HODE and 10S-HODE as standards. 8R-HODE eluted after 8S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10S-HODE eluted before 10R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5S,8R-Dihydroxyoctadeca-9Z,12Z-dienoic acid (5S,8R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8R,11S-dihydroxyoctadeca-9Z,12Z-dienoic acid (8R,11S-DiHODE) by Agaricus bisporus. 8R-Hydroperoxylinoleic acid (8R-HPODE) can be transformed to 5S,8R-DiHODE and 8R,11-DiHODE by Aspergillus spp., and 8R,13-dihydroxy-9Z,11E-dienoic acid (8R,13-DiHODE) can also be detected. We prepared racemic [5,8-2H2]5,8- and [8,11-2H2]8,11-DiHODE by oxidation and reduction as above and 8R,13S- and 8R,13R-DiHODE by oxidation of 8R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC–MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8R-HODE (>95% R), 10R-HODE (>70% R), and 5S,8R- and 8R,11S-DiHODE with high stereoselectivity (>95%), whereas 8R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8R,11S-DiHODE.

  • 28.
    Geitmann, Matthis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 332, no 2, p. 203-214Article in journal (Refereed)
    Abstract [en]

    The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.

  • 29.
    Ghafouri, Bijar
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Occupational and Environmental Medicine. Linköping University, Faculty of Health Sciences.
    Karlsson, Helen
    Linköping University, Department of Molecular and Clinical Medicine, Occupational and Environmental Medicine. Linköping University, Faculty of Health Sciences.
    Mörtstedt, Harriet
    Linköping University, Department of Molecular and Clinical Medicine. Linköping University, Faculty of Health Sciences.
    Lewander, Andreas
    Linköping University, Department of Biomedicine and Surgery. Linköping University, Faculty of Health Sciences.
    Tagesson, Christer
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Occupational and Environmental Medicine. Östergötlands Läns Landsting, Pain and Occupational Centre, Occupational and Environmental Medicine Centre.
    Lindahl, Mats
    Linköping University, Department of Molecular and Clinical Medicine, Occupational and Environmental Medicine. Linköping University, Faculty of Health Sciences.
    Note: 2,5-Dihydroxybenzoic acid instead of α-cyano-4-hydroxycinnamic acid as matrix in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for analyses of in-gel digests of silver-stained proteins: in Analytical Biochemistry(ISSN 0003-2697), vol 371, issue 1, pp 121-1232007In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 371, no 1, p. 121-123Article in journal (Other academic)
    Abstract [en]

    [No abstract available]

  • 30. Gharizadeh, B.
    et al.
    Eriksson, J.
    Nourizad, N.
    Nordstrom, Tommy
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Improvements in pyrosequencing technology by employing sequenase polymerase2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 330, no 2, p. 272-280Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.

  • 31. Gharizadeh, B.
    et al.
    Nordstrom, T.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Long-read pyrosequencing using pure 2 '-deoxyadenosine-5 '-O '-(1-thiotriphosphate) Sp-isomer2002In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 301, no 1, p. 82-90Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.

  • 32.
    Gharizadeh, Baback
    et al.
    Stanford Genome Technology Center, Stanford University, Palo Alto, USA.
    Eriksson, Jonas
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Nourizad, Nader
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Nordström, Tommy
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Nyrén, Pål
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Improvements in Pyrosequencing technology by employing Sequenase polymerase2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 330, no 2, p. 272-280Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.

  • 33. Ginya, Harumi
    et al.
    Asahina, Junko
    Yoshida, Mamiko
    Segawa, Osamu
    Asano, Tsutomu
    Ikeda, Hideo
    Hatano, Yukiko Miyashita
    Shishido, Madoka
    Johansson, Britt-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Zhou, Qin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hallberg, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Takahashi, Masaaki
    Nyberg, Fred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tajima, Hideji
    Yohda, Masafumi
    Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G)2007In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 367, no 1, p. 79-86Article in journal (Refereed)
    Abstract [en]

    We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.

  • 34.
    Gullberg, Jonas
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Jonsson, Pär
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nordström, Anders
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Sjöström, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Moritz, Thomas
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 331, no 2, p. 283-295Article in journal (Refereed)
    Abstract [en]

    The usual aim in metabolomic studies is to quantify the entire metabolome of each of a series of biological samples. To do this for complex biological matrices, e.g., plant tissues, efficient and reproducible extraction protocols must be developed. However, derivatization protocols must also be developed if GC/MS (one of the mostly widely used analytical methods for metabolomics) is involved. The aim of this study was to investigate how different chemical and physical factors (extraction solvent, derivatization reagents, and temperature) affect the extraction and derivatization of the metabolome from leaves of the plant Arabidopsis thaliana. Using design of experiment procedures, variation was systematically introduced, and the effects of this variation were analyzed using regression models. The results show that this approach allows a reliable protocol for metabolomic analysis of Arabidopsis to be determined with a relatively limited number of experiments. Following two different investigations an extraction and derivatization protocol was chosen. Further, the reproducibility of the analysis of 66 endogenous compounds was investigated, and it was shown that both hydrophilic and lipophilic compounds were detected with high reproducibility.

  • 35.
    Gustafsson, Sofia S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Vrang, Lotta
    Terelius, Ylva
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Quantification of interactions between drug leads and serum proteins by use of "binding efficiency"2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 409, no 2, p. 163-175Article in journal (Refereed)
    Abstract [en]

    To develop efficient and reliable methods for prediction of serum protein binding of drug leads, the kinetic characteristics for the interactions between selected compounds and human serum albumin and α(1)-acid glycoprotein have been explored using a surface plasmon resonance biosensor. Conventional methods for quantification of interactions (i.e., using rate constants or affinities determined on the basis of a reasonable mechanistic model) were applicable for only a few of the compounds. The affinity of a primary interaction and the contribution of lower affinity secondary interactions could be estimated for some compounds, but the affinity of many compounds could not be quantified by either of these methods. To have a quantification method that could be used for all compounds, independent of affinity and complexity of interaction mechanisms, the concept of "binding efficiency," analogous to "catalytic efficiency" used for enzymes, was developed. It allowed the quantification of the binding of compounds interacting with weak affinity and for which saturation is not reached within a concentration range where the compound is soluble or when the influence of interactions with secondary sites makes interpretations difficult. In addition, compounds with large fractional binding can be identified by this strategy and simply quantified relative to reference compounds. This approach will enable ranking and identification of structure-activity relationships of compounds with respect to their serum protein binding profile.

  • 36.
    Gutierrez Arenas, Omar
    et al.
    Department of Biochemistry and Organic Chemistry, Uppsala University.
    Danielson, U Helena
    Detection of competitive enzyme inhibition with end point progress curve data2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, no 1, p. 11-19Article in journal (Refereed)
    Abstract [en]

    A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.

  • 37.
    Gutierrez Arenas, Omar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Detection of competitive enzyme inhibition with end point progress curve data2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, no 1, p. 11-19Article in journal (Refereed)
    Abstract [en]

    A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.

  • 38.
    Gutierrez Arenas, Omar
    et al.
    Department of Biochemistry and Organic Chemistry, Uppsala University.
    Danielson, U Helena
    Sensitivity analysis and error structure of progress curves2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Both the sensitivity of the monitored signal in progress curves to variations in enzyme concentration and the standard deviation of this signal were analyzed as a function of the proportion of transformed substrate. Three enzymes catalyzing essentially irreversible reactions were used as model systems: HIV-1 protease, glutathione reductase, and glutathione transferase. For all enzymes analyzed, the sensitivity was maximal when 60-80% of the substrate had been transformed. The standard deviation of reaction progress curve data replicates was also maximal at these substrate conversion levels, a result that was attributed to the influence of the sensitivity to random dispersion of the enzyme concentration. On this basis, we developed a model for the standard deviation of reaction progress curves that gave a good description of the experimental data and efficiently reduced the heteroscedasticity of residuals in a weighted fit of progress curves. This standard deviation model can be used for obtaining more efficient parameter estimates, to simulate noise in Monte Carlo procedures, and to delineate detection limits of enzyme inhibition. The transient increases in the sensitivity and in the standard deviation in progress curves are proposed to be features common to most enzymatic assays.

  • 39.
    Gutierrez Arenas, Omar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Sensitivity analysis and error structure of progress curves2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Both the sensitivity of the monitored signal in progress curves to variations in enzyme concentration and the standard deviation of this signal were analyzed as a function of the proportion of transformed substrate. Three enzymes catalyzing essentially irreversible reactions were used as model systems: HIV-1 protease, glutathione reductase, and glutathione transferase. For all enzymes analyzed, the sensitivity was maximal when 60-80% of the substrate had been transformed. The standard deviation of reaction progress curve data replicates was also maximal at these substrate conversion levels, a result that was attributed to the influence of the sensitivity to random dispersion of the enzyme concentration. On this basis, we developed a model for the standard deviation of reaction progress curves that gave a good description of the experimental data and efficiently reduced the heteroscedasticity of residuals in a weighted fit of progress curves. This standard deviation model can be used for obtaining more efficient parameter estimates, to simulate noise in Monte Carlo procedures, and to delineate detection limits of enzyme inhibition. The transient increases in the sensitivity and in the standard deviation in progress curves are proposed to be features common to most enzymatic assays.

  • 40.
    Göransson, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Broussalis, Adriana M
    Claeson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Expression of Viola cyclotides by liquid chromatography-mass spectrometry and tandem mass spectrometry sequencing of intercysteine loops after introduction of charges and cleavage sites by aminoethylation2003In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 318, no 1, p. 107-117Article in journal (Refereed)
    Abstract [en]

    The expression of cyclotides—macrocyclic plant peptides—was profiled in six violets, Viola cotyledon, V. biflora, V. arvensis,V. tricolor, V. riviniana, and V. odorata, by LC-MS. All were found to express notably complex mixtures, with single speciescontaining >50 cyclotides. To facilitate their sequencing by MS-MS, an analytical strategy is presented involving aminoethylation ofcysteines. This overcomes a number of problems intimately associated with the cyclotide core structure—that is, their joined N and Ctermini, disulfide knot, and low or clustered content of positively charged amino acids and enzymatic cleavage sites. As a result,charges as well as cleavage sites are introduced at the most conserved part of their sequence, the cysteines. Combined with trypticdigestion, all intercysteine loops are then of suitable size and charge for MS-MS sequencing. The utility of this strategy is shown bythe sequencing of two novel cyclotides isolated from V. cotyledon; vico A (cyclo-(AESCVYIPCFTGIAGCSCKNKVCYYNGSIPC)) and vico B(cyclo-(AESCVYIPCITGIAGCSCKNKVCYYNGSIPC)); their complete sequence could be determined bynanospray MS-MS. The strategy for converting conserved cysteines to enzymatic cleavage sites might also benefit the study of otherpeptides and proteins displaying similar structural problems for MS analysis.

  • 41.
    Halldén, Christer
    et al.
    Hilleshög AB, Landskrona.
    Nilsson, N. O.
    Hjerdin, A.
    Rading, I. M.
    A gel electrophoresis system adapted for large-scale molecular marker analysis1996In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 242, no 1, p. 145-7Article in journal (Refereed)
  • 42.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kempka, Martin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Sjödahl, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, no 2, p. 167-172Article in journal (Refereed)
    Abstract [en]

     A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

  • 43.
    Hedman, Johannes
    et al.
    Department of Applied Microbiology, Lund University, Sweden.
    Nordgaard, Anders
    Swedish National Laboratory of Forencis Sciences, Linkoping, Sweden.
    Dufva, Charlotte
    National Laboratory of Forensic Sciences, Linkoping, Sweden.
    Rasmusson, Birgitta
    National Laboratory of Forensic Sciences, Linkoping, Sweden.
    Ansell, Ricky
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics. Linköping University, The Institute of Technology.
    Rådström, Peter
    Department of Applied Microbiology, Lund University, Sweden.
    Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis2010In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 405, p. 192-200Article in journal (Refereed)
    Abstract [en]

    The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substancespresent in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene salivastains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNApolymerase–buffer systems (Hedman et al., BioTechniques 47 (2009) 951–958). Here we show thatblending inhibitor-resistant DNA polymerase–buffer systems further increases the success rate of PCRfor various types of real crime scene samples showing inhibition. For 34 of 42 ‘‘inhibited” crime scenestains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq HotStart and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmedby analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerasesused separately for all tested sample types. When used separately, the performance of the DNApolymerases varied depending on the nature of the sample. The superiority of the blend is discussed interms of complementary effects and synergy between the DNA polymerase–buffer systems.

  • 44.
    Helmfors, Henrik
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eriksson, Jonas
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides2015In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 484, p. 136-142Article in journal (Refereed)
    Abstract [en]

    An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.

  • 45.
    Jacksén, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Evaluation of 2,6-dihydroxyacetophenone as matrix-assisted laser desorption/ionization matrix for analysis of hydrophobic proteins and peptides2012In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 425, no 1, p. 18-20Article in journal (Refereed)
    Abstract [en]

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for analysis of macromolecules like peptides and proteins. The analysis procedure is generally simple but must be adapted to the characteristics of the analytes. Therefore, specific matrices suitable for, e.g., hydrophobic proteins and peptides that are difficult to analyze would be preferable in order to optimize the outcome. In the present work, 2,6-dihydroxyacetophenone (DHAP) was shown to be beneficial in comparison to DHB for intact bacteriorhodopsin (BR) as well as for chemically digested BR.

  • 46.
    Joda, Hamdi
    et al.
    Universitat Rovira i Virgili, Tarragona, Spain.
    Beni, Valerio
    INTERFIBIO Research Group, Departament d’Enginyeria Quimica, Universitat Rovira i Virgili, Tarragona, Spain.
    Katakis, Ioanis
    Universitat Rovira i Virgili, Tarragona, Spain.
    O´Sullivan, Ciara K.
    Universitat Rovira i Virgili, Tarragona, Spain.
    DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection2015In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 474, p. 66-68Article in journal (Refereed)
    Abstract [en]

    We report on a simple approach to enhance solid-phase hybridization-based single base mismatch discrimination at high ionic strength based on the deliberate insertion of a natural DNA base mismatch in the surface-tethered probe. A large drop in hybridization signal of single base mismatched alleles using the designed probe as compared with the conventional probe, from 80% to less than 25% of the signal obtained with the fully complementary, non-mutation-containing sequence, when using colorimetric detection was further improved to 20% when using electrochemical detection, attributable to a difference of spacing of immobilized probes. Finally, the designed probe was used for the electrochemical detection of the DQA1*05:05 allele amplified from real human blood samples.

  • 47.
    Jungar, Christina
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biotechnology. Linköping University, Faculty of Science & Engineering.
    Strandh, M
    University of Kalmar, Kalmar, Sweden.
    Ohlson, S
    University of Kalmar, Kalmar, Sweden.
    Mandenius, Carl-Fredrik
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biotechnology.
    Analysis of carbohydrates using liquid chromatography-surface plasmon resonance immunosensing systems2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 281, no 2, p. 151-158Article in journal (Refereed)
    Abstract [en]

    An immunosensing system based on surface plasmon resonance (SPR) was used for on-line detection and characterization of carbohydrate molecules separated by high-performance liquid chromatography. These analytes, with or without serum, were continuously separated and analyzed in the combined liquid chromatography-surface plasmon resonance (LC-SPR) system. By using weak and readily reversible monoclonal antibodies, the SPR system allowed specific on-line monitoring of the substances. To increase the specificity of the immunosensor, nonrelevant antibodies were used as reference in a serial flow cell. The sensitivity of the LC-SPR system was dependent on molecular weight of the carbohydrate, affinity of binding, and design of the sensor. (C) 2000 Academic Press.

  • 48. Karlstrom, A.
    et al.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Dual labeling of a binding protein allows for specific fluorescence detection of native protein2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 295, no 1, p. 22-30Article in journal (Refereed)
    Abstract [en]

    Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal. protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn(23)Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-LAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl) amino) hexanoate (NBD-X, SE), respectively. Biosensor analysis of Purified doubly labeled protein showed that high-affinity binding to the Fe region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of FC3(1)) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc(3(1)), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

  • 49. Khihon Rokhas, Maria
    et al.
    Liljestrand Rönn, Johanna
    Wiklund, Christer
    Stockholm University, Faculty of Science, Department of Zoology.
    Emmer, Åsa
    Analysis of butterfly reproductive proteins using capillary electrophoresis and mass spectrometry2019In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 566, p. 23-26Article in journal (Refereed)
    Abstract [en]

    A method for analysis of proteins from spermatophores transferred from male to female Pieris napi butterflies during mating has been developed. The proteins were solubilized from the dissected spermatophores using different solubilization agents (water, methanol, acetonitrile and hexafluoroisopropanol). Capillary electrophoresis (CE) analysis was performed using an acidic background electrolyte containing a fluorosurfactant to avoid protein-wall adsorption, and to increase separation performance. The samples were also analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), in a lower m/z range (1000-6000) and a higher m/z range (6000-12000). Solubilization with different solvents and the use of alternative matrices gave partly complementary profiles.

  • 50. Kjellander, Marcus
    et al.
    Mazari, Aslam M. A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Boman, Mats
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry. Uppsala University, Sweden.
    Johansson, Gunnar
    Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability2014In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, p. 59-63Article in journal (Refereed)
    Abstract [en]

    The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via c-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

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