Quantitative gel electrophoresis: New records in precision by elaborated staining and detection protocols

Abstract

Gel electrophoresis (GE) is a very common analytical technique for proteome research and protein analysis. Despite being developed decades ago, there is still a considerable need to improve its precision. Using the fluorescence of Colloidal Coomassie Blue -stained proteins in near-infrared (NIR), the major error source caused by the unpredictable background staining is strongly reduced. This result was generalized for various types of detectors. Since GE is a multi-step procedure, standardization of every single step is required. After detailed analysis of all steps, the staining and destaining were identified as the major source of the remaining variation. By employing standardized protocols, pooled percent relative standard deviations of 1.2-3.1% for band intensities were achieved for one-dimensional separations in repetitive experiments. The analysis of variance suggests that the same batch of staining solution should be used for gels of one experimental series to minimize day-to-day variation and to obtain high precision.

Capillary gel electrophoresis for precise protein quantitation

“…On‐column detection with an UV‐Vis detector reduces manual work and related errors. Even though the quantitation in SDS‐PAGE was meanwhile improved to a precision corresponding to RSD% values up to 3% by using standard operating procedures (SOPs) , the handling in CE is significantly easier. However, a higher protein concentration (>0.1 mg/mL) needs to be injected into the CE system than used in SDS‐PAGE (<0.1 mg/mL).…”

A ‘green’ approach to fixing polyacrylamide gels

“…Gel fixation after the electrophoretic resolution of proteoforms serves to precipitate the species within the gel matrix at their point of resolution via an acidic wash, which also enhances their subsequent interaction with stains by removing SDS [5,6]. Currently, a methanol or ethanol and acetic acid combination is routinely used as a fixative, particularly prior to staining with colloidal Coomassie Brilliant Blue (cCBB) [4,[7][8][9][10][11][12]. Unfortunately, when methanol is used for gel fixation following SDS-PAGE, it induces methylation at the side chains of aspartate and glutamate; substitution with ethanol results in ethylation of these same residues.…”