Electrophoretic artifacts arising from the use of thiol‐containing reagents

Abstract

Thiol reagents migrate as a curtain behind the salt front when loaded with the sample solution onto disc-electrophoresis gels. In immobilized pH gradients (IPG) the same compounds are driven by electrophoresis and electroosmosis from the alkaline to the neutral and acidic regions of the gradients. In either case, a dose-dependent sideways spreading results in spurious reduction between adjacent lanes if samples with and without reducing agent are loaded side by side.

Other than IPG‐DALT: 2‐DE variants

“…Inclusion of DTT in the reswelling solution during in‐gel rehydration procedures for sample loading appears insufficient to prevent cysteine (re)oxidation in alkaline proteins. With time DTT is driven by electrophoresis and electroosmosis away from the alkaline region of a wide range IPG, where its action would be needed, to concentrate in the neutral and acidic regions 44. With individual alkaline proteins, (re)oxidation is made evident by spurious spots due to oligomeric assemblies via scrambled disulfide bridges 45.…”

“…Established “all‐purpose” protocols currently call for CHAPS as the detergent of choice as having the lowest micelle weight (6150 Da) and aggregation number (10); equilibration between 1d and 2d is in urea‐SDS for less than half an hour. This procedure is undemanding but − even in the best laboratories, as seen in many published maps − its implementation sometimes results in striped “salt fronts”, mostly over the 4−6 pH range that collects electroendoosmotic flow from wide pH ranges 44. This interference also sets a limit to the width of the 1d gel strip as affecting the amount of residual detergent the system has to buffer.…”