Reproducible protein analysis by CE using linear polyacrylamide‐coated capillaries and hydrochloric acid rinsing

Abstract

Hydrochloric acid was investigated as a rinsing reagent to remove adsorbed proteins from linear polyacrylamide-coated capillaries for electrophoresis. Three model proteins were used, namely cytochrome c as a basic protein, beta-lactoglobulin as an acidic protein, and beta-casein as a more easily denaturing protein. In order to regenerate capillary surfaces, they have been rinsed for 5 min with 2 M hydrochloric acid, 5 min with water, and then 30 min with buffer after every tenth run. It was found important to perform this regeneration procedure on time. The obtained results show good repeatability of the apparent EOF mobility with percentage RSDs below 3% (n = 60) in various cases. These good results were mainly confirmed in long-term series with more than 200 runs each. Only very high concentrations (175 microM) of beta-lactoglobulin and beta-casein at pH 3.5 gave RSD% values above 5%. For these conditions, the further test of 85% m/m phosphoric acid as rinsing reagent showed a good repeatability of the apparent EOF mobilities as well.

A quantitative CE method to analyse tau protein isoforms using coated fused silica capillaries

“…The performances were slightly better with phosphate buffer than with other buffers. This is consistent with previous studies showing that phosphate may interact with proteins and stabilise their tertiary structure [48], but also may efficiently screen the silica surface, thereby reducing the adsorption of proteins [49]. Thus, as listed in Table 2, an efficient separation of Tau-352 and Tau-441 could be achieved in less than 15 min with 50 mM phosphate buffer pH 3.0 (Fig.…”

Capillary electrophoresis analysis of biofluids with a focus on less commonly analyzed matrices

“…A variety of post-run washing procedures optimized for the contaminants and surfaces involved have been developed [32][33][34]. A general limitation of CE is the relatively high limit of detection usually obtained with UV detection.…”

Long‐term precision in capillary isoelectric focusing for protein analysis

“…The proteins thus modify the pH gradient that is established by the ampholyte [6]. This protein property may change over time [11]. The resulting change in migration behavior cannot be compensated for by the internal standard that does not show this property.…”

“…Among these, alkaline rinsing solutions or solutions containing SDS, which are very useful in CZE, are not suitable for CIEF, because they may damage or irreversibly change the surface properties of the usually employed polyacrylamide-coated capillaries [5,9]. However, the use of hydrochloric acid has also been successfully applied to polyacrylamide-coated capillaries to improve the reproducibility of migration times and peak areas in protein analysis [10,11]. Another rinsing reagent, namely phosphoric acid 85% m/m also proved to be effective, especially in the case of highly concentrated protein samples or the more easily defolding ones [11].…”

“…However, the use of hydrochloric acid has also been successfully applied to polyacrylamide-coated capillaries to improve the reproducibility of migration times and peak areas in protein analysis [10,11]. Another rinsing reagent, namely phosphoric acid 85% m/m also proved to be effective, especially in the case of highly concentrated protein samples or the more easily defolding ones [11]. Several rinsing and storage procedures for coated capillaries, such as water rinsing, water rinsing followed by N 2 rinsing before capillary storage, or storage of the capillary in distilled water were also described [6,10].…”