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  • 1.
    Bohlin, Maria
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Capillary electrophoresis of b2-glycoprotein I: Method Development for Binding Studies2009Inngår i: / [ed] Lars G. Blomberg, Niels H. H. Heegaard, 2009Konferansepaper (Annet (populærvitenskap, debatt, mm))
  • 2.
    Bohlin, Maria
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Capillary electrophoresis of beta2-glycoprotein I: Method development and binding studies2006Licentiatavhandling, monografi (Annet vitenskapelig)
  • 3.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Affinity studies of beta-2-glycoprotein I using capillary electrophoresis2010Inngår i: / [ed] Lars G. Blomberg, Niels H. H. Heegaard, 2010Konferansepaper (Fagfellevurdert)
    Abstract

    Beta2-glycoprotein I (b2gpI), also known as apolipoprotein H, is a plasma protein which is involved in the blood coagulation cascade. It binds negatively charged substances such as heparin, DNA, and anionic phospholipids. A number of functions of b2gpI have been proposed, however, the precise function is still not entirely known. Circulating autoantibodies against b2gpI are associated with increased risk of thrombotic events, such as thrombosis and reoccurring fetal loss. It is therefore of interest to functionally characterize b2gpI including the influence of anti-b2gpI autoantibodies on the ligand binding behavior of the protein. The characterization of interactions between biological molecules may be accomplished by capillary electrophoresis under non-denaturing conditions, without the need for immobilization. To avoid charge dependent analyte adsorption to the negative charges of the capillary wall we found the pH hysteresis effect of silica very useful. An acidic pretreatment of the capillary made it possible to perform a subsequent analysis at neutral pH. We were able to perform binding studies between b2gpI and heparin and monosaccharides at different ionic strengths and temperatures in a simple way. We could also study the effect of mildly denaturing conditions on the binding to the different ligands simply by adding sodium dodecyl sulfate (SDS), urea and ACN to the background electrolyte.

    The approach is simple, fast and automatic. The ionic strength, temperature and other parameters such as denaturing agents could easily be changed to characterize the binding between b2gpI and different ligands.

  • 4.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Affinity studies of beta2-glycoprotein I using capillary electrophoresis2010Inngår i: / [ed] Lars G. Blomberg, Niels Heegaard, 2010Konferansepaper (Fagfellevurdert)
    Abstract

    beta2-glycoprotein I (b2gpI), also known as apolipoprotein H, is a plasma protein which is involved in the blood coagulation cascade. It binds negatively charged substances such as heparin, DNA, and anionic phospholipids. A number of functions of b2gpI have been proposed, however, the precise function is still not entirely known. Circulating autoantibodies against b2gpI are associated with increased risk of thrombotic events, such as thrombosis and reoccurring fetal loss. It is therefore of interest to functionally characterize b2gpI including the influence of anti-b2gpI autoantibodies on the ligand binding behavior of the protein. The characterization of interactions between biological molecules may be accomplished by capillary electrophoresis under non-denaturing conditions, without the need for immobilization. To avoid charge dependent analyte adsorption to the negative charges of the capillary wall we found the pH hysteresis effect of silica very useful. An acidic pretreatment of the capillary made it possible to perform a subsequent analysis at neutral pH. We were able to perform binding studies between b2gpI and heparin at different ionic strengths and temperatures in a simple way. We could also study the effect of mildly denaturing conditions on the binding to the ligand simply by adding sodium dodecyl sulfate (SDS), urea and ACN to the background electrolyte. The approach is simple, fast and automatic

  • 5.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Affinity-CE with phospholipid and carbohydrate ligands: Binding between heparin and anionic liposomes and the anticoagulant protein, beta-2-glycoprotein I2004Inngår i: / [ed] E. Kogutowska, L. Blomberg, N. H. H. Heegaard, 2004Konferansepaper (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    Binding studies executed by capillary electrophoresis (CE) benefit from flexibility in experimental set-up, wide applicability, and low consumption of reagents. Importantly, the high resolution inherent in CE allows complex mixtures and binding resulting in only small migration changes to be characterized quantitatively. One specific problem, however, that may be encountered when applying CE for the analysis of binding interactions of proteins is the need to avoid interactions other than those between the analyte and the ligand. Thus, protein-wall interactions in unmodified fused-silica capillaries used at neutral pH often invalidate analyses of protein binding. This problem is especially pronounced with basic proteins and proteins containing exposed patches of positive charge. Ways to overcome this are to neutralize the immobilized wall charges e.g. by different wall coatings and/or buffer additives, or by using the pH hysteresis effect, i.e., the slow deprotonation at neutral pH of low pH-treated fused silica. We illustrate the use of the pH-hysteresis effect to avoid adsorption problems in the analysis of a phospholipid- and heparin-binding anticoagulant protein, beta-2-glycoprotein under physiological pH conditions. The approach was useful for characterizing protein-heparin and protein-liposome binding. This will be the basis of using CE methods to evaluate the influence of human thrombogenic autoantibodies against this protein on such interactions and the approach will be also be a useful means to measure the binding activity of different domains and structurally modified variants of this protein and other proteins that suffer from a tendency to stick to fused silica

  • 6.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Capillary electrophoresis-based analysis of phospholipid- and glycosaminoglycan-binding by human beta-2-glycoprotein I2004Inngår i: / [ed] E. Kogutowska, L. Blomberg, N. H. H. Heegaard, 2004Konferansepaper (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    Human beta-2-glycoprotein I (b2gpI) is a phospholipid- and heparin-binding plasma glycoprotein involved in autoimmune diseases characterized by blood clotting disturbances (thrombosis) together with the occurrence of autoantibodies against b2gpI. With the final goal of assessing autoantibody influence on binding interactions of b2gpI we have studied the development of capillary electrophoresis (CE)-based assays for interactions of negatively charged ligands with b2gpI. Binding studies executed by capillary electrophoresis benefit from flexibility in experimental set-up, wide applicability, and low consumption of reagents.

    Importantly, the high resolution inherent in CE allows complex mixtures and binding resulting in only small migration changes to be characterized quantitatively. One specific problem, however, that may be encountered when applying CE for the analysis of binding interactions of proteins is the need to avoid interactions other than those between the analyte and the ligand. Thus, protein-wall interactions in unmodified fused-silica capillaries used at neutral pH often invalidate analyses of protein binding. This problem is especially pronounced with basic proteins and proteins containing exposed patches of positive charge. In the development of suitable conditions for analysis at neutral pH of b2gpI we found the pH hysteresis behavior of fused silica surfaces useful since the protonated surface after an acid pre-wash counteracted protein adsorption efficiently in contrast to more laborious procedures including acrylamide/dimethylacrylamide coatings that did not permit analysis of this particular protein. This approach made quantitative studies of heparin-b2gpI interactions possible and the principle was shown also to work for detection of b2gpI binding to anionic phospholipids. Utilizing the pH hysteresis effect may be a simple solution to the adsorption problems often encountered in analyses of proteins by CE.

  • 7.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Use of Dimethylacrylamide Coated Capillaries for the Study of beta2-Glycoprotein I with Affinity Capillary Electrophoresis2003Inngår i: / [ed] Lars G. Blomberg, Niels H. H. Heegaard, 2003Konferansepaper (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    The purpose of this work is to develop a solution method for examining interactions of human beta2-Glycoprotein I (b2gpI) with antibodies and natural ligands under nondenaturing conditions. In the approach, a physiological pH was applied because our intention was to study the interactions between b2gpI and antibodies and the protein must thus be kept in its native state. Preliminary experiments have shown that the recovery of the protein, when analyzed in an uncoated capillary, is poor. At physiological pH the protein is positively charged and most likely it was adsorbed at the negatively charged capillary wall in these experiments. Therefore dimethylacrylamide (DMA) coated capillaries1 are used and analyses could be performed with acceptable recovery. Interaction studies are performed simply by adding the antibody or ligand to the running buffer. The method showed good repeatability and efficiency

  • 8.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Heegaard, N.H.H.
    Utilizing the pH Hysteresis Effect for Versatile and Simple Electrophoretic Analysis of Proteins in Bare Fused-Silica Capillaries2005Inngår i: Electrophoresis, 26 (2005) 4043-4049Artikkel i tidsskrift (Fagfellevurdert)
  • 9.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Olsson, Ola
    Heegaard, Niels H.H
    Structure-activity studies of human beta2-glycoprotein I using capillary electrophoresis2011Konferansepaper (Fagfellevurdert)
    Abstract

    We have investigated various modes of CE to evaluate the interaction between beta2-glycoprotein I (b2gpI) and a number of anionic ligands to contribute to the elucidation of the structure-function relationship of b2gpI. b2gpI is a plasma protein which is involved in the blood coagulation cascade under normal, physiological conditions, however, its precise function is undefined. It is also involved in pathological conditions such as the so-called anti-phospholipid syndrome, where anti-b2gpI autoantibodies induce a prothrombotic state. Therefore, functional characterization of b2gpI under near physiological conditions is of interest.

    To avoid charge-dependent analyte adsorption to the inner surface of the capillary wall, we have utilized the pH hysteresis effect, where an acidic pretreatment of the capillary made it possible to perform subsequent CE analyses of b2gpI at neutral pH.

    The interaction between b2gpI and the anionic ligand heparin was studied with migration shift ACE, where the ionic strength, temperature and conformation of b2gpI were easily varied. The interaction between b2gpI and phosphatidylcholine/phosphatidylserine liposomes are subject to an ongoing investigation by means of migration shift ACE, frontal analysis CE, partial filling CE and pre-equilibration partial filling ACE.

    We conclude that differential, but relatively low binding affinities that are highly dependent on electrostatic interactions and on a preserved conformation of the protein, characterize its interactions with ligands that in vivo will be present in multiple copies on e.g. cell surfaces. The CE procedure for this study is simple, fast and automatic and quantitative binding affinity parameters are conveniently obtained using small amounts of biological materials.

  • 10.
    Bohlin, Maria E.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands2011Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The final goal of this study is to establish a microscale analysis method that allows solution phase characterization of interactions between β2-glycoprotein I (β2gpI) and some of its ligands. Human β2gpI is a phospholipid- and heparin-binding plasma glycoprotein. The physiological role of the protein in normal blood coagulation is not entirely known, nor is its role in autoimmune diseases characterized by blood clotting disturbances (thrombosis). Quantitative binding data of β2gpI interactions with some of its ligands may help elucidating the mechanisms behind these diseases and in the development of new approaches for diagnostics, prevention, and therapy.

    In this thesis, capillary electrophoresis (CE) was used as methodological platform for the interaction studies. The analysis of peptides and proteins by CE is desirable due to low sample consumption, possibilities for non-denaturing and highly effective separations. The first objective of this thesis was to find an approach to prevent charge dependent adsorption of β2gpI to the inner surface of the capillaries. Analyte adsorption at the negatively charged inner surface of fused silica capillaries is detrimental to interaction analyses. This phenomenon is especially pronounced in the analysis of basic proteins and proteins containing exposed positively charged domains, such as β2gpI. A new strategy to suppress these solute-wall interactions was devised, investigated and optimized. This strategy exploits the pH hysteresis behavior of fused silica surfaces, by simply performing an acidic pretreatment of the capillary. The results in this thesis show that the acidic pretreatment efficiently prevents protein adsorption.

  • 11.
    Bohlin, Maria E
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Blomberg, Lars G.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Heegaard, Niels H H
    Statens Serum Institut, Copenhagen.
    Effects of ionic strength, temperature and conformation on affinity interactions of β2-glycoprotein I monitored by capillary electrophoresis2011Inngår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, s. 728-737Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have used CE to evaluate the interaction between β2-glycoprotein I (β2gpI) and heparin. β2gpI is a human plasma protein involved in the blood coagulation cascade. It is of interest to functionally characterize the interactions of β2gpI because the exact function is not entirely known and because circulating autoantibodies against β2gpI are associated with an increased risk of thrombotic events.

     

    The effect of the ionic strength, temperature, and conformation of the protein on the interaction between β2gpI and heparin has been studied. The CE procedure for this study is simple, fast and automatic. β2gpI and heparin were allowed to interact during electrophoresis at different ionic strength buffers and at different capillary temperatures. To mimic perturbation of the conformation of β2gpI, different denaturing agents (SDS, ACN and urea) were added to the background electrolyte. While simple 1:1 binding isotherms were obtained at 22 °C the data strongly suggests that at physiological temperature the binding stoichiometry is not 1:1 and/or that cooperative interactions begin to play a role. We found that (i) the KD values differed by a factor of 60 at the ionic strengths studied (ii) β2gpI was resistant to denaturation with SDS and ACN, but was partially denatured by urea and (iii) the KD for the β2gpI-heparin interaction in the presence of urea was 10 times higher than the KD determined at the same conditions without urea added. Therefore, we conclude that the interaction between β2gpI and heparin is dependent on electrostatic interactions and on the conformation of β2gpI. 

  • 12.
    Bohlin, Maria E.
    et al.
    Karlstads universitet, Institutionen för kemi.
    Blomberg, Lars G.
    Karlstads universitet, Institutionen för kemi.
    Heegard, Niels H.H.
    Department of Autoimmunology, Statens Serum Institut, Copenhagen.
    Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of protein in bare fused-silica capillaries2005Inngår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, nr 21, s. 4043-4049Artikkel i tidsskrift (Fagfellevurdert)
  • 13.
    Bohlin, Maria E.
    et al.
    Karlstads universitet, Institutionen för kemi.
    Kogutowska, Ewa
    Department of Autoimmunology, Statens Serum Institut, Copenhagen.
    Blomberg, Lars G.
    Karlstads universitet, Institutionen för kemi.
    Heegaard, Niels H.H.
    Department of Autoimmunology, Statens Serum Institut, Copenhagen.
    Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human β2-glycoprotein I2004Inngår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1059, s. 215-222Artikkel i tidsskrift (Fagfellevurdert)
  • 14.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Johannesson, Ida
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Carlsson, Gunilla
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Heegaard, Nils H H
    Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark.
    Blomberg, Lars G.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment2012Inngår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, nr 12, s. 1695-1702Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Sample adsorption to the inner surface of fused silica capillaries is a common problem in

    CE when analyzingmacromolecules and is harmful to the analysis. We previously utilized

    the pH hysteresis effect of fused silica to facilitate electrophoresis of the strongly adsorbing

    protein β2gpI in plain-fused silica capillaries at neutral pH. In the present paper, the

    effect of different pretreatments of the capillary on the adsorption of the β2-glycoprotein

    I has been investigated using electroosmosis markers, SDS mobilization, and imaging

    based on indirect immunofluorescence microscopy for direct visualization. The amount

    of β2gpI adsorbed on the surface was probed using all these independent techniques after

    electrophoresis at neutral pH on capillaries pretreated with HCl, background electrolyte

    (BGE), and NaOH. BGE pretreatment was included as a positive control. We found that

    80% or more of the starting material was adsorbed to the inner surface of the silica

    capillaries during electrophoresis after pretreatment with only BGE or with NaOH, but

    after acidic pretreatment the loss was consistently less than 20%. NaOH most efficiently

    removes adsorbed protein between runs. A theoretical calculation of the pH change of

    the BGE showed that electrolysis affects the pH more than the deprotonation of silanols

    during electrophoresis. We conclude that acidic pretreatment of fused silica capillaries

    diminishes adsorption of β2gpI by decreasing charge-dependent wall adsorption.

     

  • 15.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Kogutowska, E.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Heegaard, N.H.H
    Capillary electrophoresis-based analysis of phospholipid- and glucosaminoglycan-binding by human β2-glycoprotein I2004Inngår i: J. Chromatogr. A, 1059 (2004) 215-222Artikkel i tidsskrift (Fagfellevurdert)
  • 16.
    Bohlin, Maria
    et al.
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Öhman, Marcus
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Hamberg, M.
    Blomberg, Lars G
    Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
    Separation of conjugated trienoic fatty acid isomers by micellar electrokinetic chromatographic2003Inngår i: J. Chromatogr. A, 985 (2003) 471-478Artikkel i tidsskrift (Fagfellevurdert)
1 - 16 of 16
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  • en-US
  • fi-FI
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