Long‐term precision in capillary isoelectric focusing for protein analysis

Abstract

In order to improve precision in protein analysis, a new rinsing procedure with 3M hydrochloric acid was investigated for linear polyacrylamide-coated capillaries used in isoelectric focusing. After each run the capillaries were rinsed with hydrochloric acid for 5 min, followed by water for 20 min (Deltap = 1030 mbar each). Myoglobin, beta-lactoglobulin, and ovalbumin were used as model proteins; the pH gradient was provided by Pharmalyte (pH 3-10). The resulting method was already successful in avoiding capillary blockages, even for long-series protein measurements. Further improvements in precision have been obtained by avoiding a complete standstill of liquid within the capillary when the separation system was idle. Pressure (Deltap = 300 mbar or more) and high voltage (30 kV) were therefore also applied during storage within a measurement series. The reproducibility of migration time and peak area are further improved with RSD% less than 10% in a long-term measurement (n = 86).

Capillary isoelectric focusing coupled offline to matrix assisted laser desorption/ionization mass spectrometry

“…However, the reproducibility of proteins would likely be less due to stronger surface interactions with the capillary wall [11]. It can be seen in the plot that the pH gradient does not exhibit uniform linearity over the entire range, and thus care must be taken in determining experimental pIs [12]. The elution times of the standard proteins was determined by MALDI-MS detection after correcting for the 4.1 minute delay between absorbance detection and fraction collection.…”

Precision in affinity capillary electrophoresis for drug–protein binding studies

“…Human serum albumin (99% agarose gel electrophoresis, pI 4.70 [23], M r 66.48 kDa), bovine serum albumin (98% agarose gel electrophoresis, pI 4.70 [20], M r 66.00 kDa), ␤-lactoglubolin (80% bovine milk, pI: 4.83-5.40 [31], M r 18.40 kDa), l-tryptophan (Trp, 98% TLC, M r 204.23) and warfarin (War, 98%, M r 308.33) were purchased from Sigma-Aldrich (Steinheim, Germany). Quercetin (Qu, 99% HPLC, M r 338.27) was obtained from Fluka (Buchs SG, Switzerland).…”

Fast investigations from biological matrices using CE – Test of a blood–brain barrier model

“…Suitable rinsing procedures are therefore crucial [52]. They may require some additional effort but can imposingly improve precision in CE, especially when proteins are involved [53]. We tested a mixture of equal parts of running buffer (60 mM borate buffer, 200 mM SDS) and acetonitrile according to Ref.…”