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Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0001-7235-0905
Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark.
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2012 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, no 12, 1695-1702 p.Article in journal (Refereed) Published
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.

 

Place, publisher, year, edition, pages
Weinheim, Germany: John Wiley & Sons, 2012. Vol. 33, no 12, 1695-1702 p.
Keyword [en]
Acidic pretreatment / Capillary electrophoresis / pH-hysteresis effect / Protein adsorption
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:kau:diva-30939DOI: 10.1002/elps.201100592ISI: 000305792500003PubMedID: 22674218OAI: oai:DiVA.org:kau-30939DiVA: diva2:689081
Available from: 2014-01-20 Created: 2014-01-20 Last updated: 2016-02-12Bibliographically approved
In thesis
1. Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
Open this publication in new window or tab >>Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Karlstad: Karlstad University, 2011. 77 p.
Series
Karlstad University Studies, ISSN 1403-8099 ; 2011:48
Keyword
Capillary Electrophoresis, β2-glycoprotein I, acidic pretreatment, pH hysteresis effect, Affinity Capillary Electrophoresis
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-8277 (URN)978-91-7063-383-6 (ISBN)
Public defence
2011-10-28, Nyquistsalen, 9C 203, Karlstads Universitet, Karlstad, 10:15 (English)
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Supervisors
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Papper 4 Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment ingick som manuskript i avhandlingen, nu publicerad.

Available from: 2011-10-07 Created: 2011-09-19 Last updated: 2016-02-12Bibliographically approved

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