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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
Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.ORCID-id: 0000-0001-7235-0905
Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark.
Visa övriga samt affilieringar
2012 (Engelska)Ingår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, nr 12, s. 1695-1702Artikel i tidskrift (Refereegranskat) 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.

 

Ort, förlag, år, upplaga, sidor
Weinheim, Germany: John Wiley & Sons, 2012. Vol. 33, nr 12, s. 1695-1702
Nyckelord [en]
Acidic pretreatment / Capillary electrophoresis / pH-hysteresis effect / Protein adsorption
Nationell ämneskategori
Kemi
Forskningsämne
Kemi
Identifikatorer
URN: urn:nbn:se:kau:diva-30939DOI: 10.1002/elps.201100592ISI: 000305792500003PubMedID: 22674218OAI: oai:DiVA.org:kau-30939DiVA, id: diva2:689081
Tillgänglig från: 2014-01-20 Skapad: 2014-01-20 Senast uppdaterad: 2019-09-19Bibliografiskt granskad
Ingår i avhandling
1. Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
Öppna denna publikation i ny flik eller fönster >>Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Karlstad: Karlstad University, 2011. s. 77
Serie
Karlstad University Studies, ISSN 1403-8099 ; 2011:48
Nyckelord
Capillary Electrophoresis, β2-glycoprotein I, acidic pretreatment, pH hysteresis effect, Affinity Capillary Electrophoresis
Nationell ämneskategori
Analytisk kemi
Forskningsämne
Kemi
Identifikatorer
urn:nbn:se:kau:diva-8277 (URN)978-91-7063-383-6 (ISBN)
Disputation
2011-10-28, Nyquistsalen, 9C 203, Karlstads Universitet, Karlstad, 10:15 (Engelska)
Opponent
Handledare
Anmärkning

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.

Tillgänglig från: 2011-10-07 Skapad: 2011-09-19 Senast uppdaterad: 2016-02-12Bibliografiskt granskad

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Förlagets fulltextPubMedhttp://onlinelibrary.wiley.com/doi/10.1002/elps.201100592/pdf

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Bohlin, MariaCarlsson, GunillaBlomberg, Lars G.

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