Simultaneous Reduction and Alkylation of Protein Disulfides in a Centrifugal Ultrafiltration Device Prior to Two-Dimensional Gel Electrophoresis

Abstract

Reduction and alkylation of protein disulfides prior to IEF, when performed directly in a centrifugal ultrafiltration device, provides an effective means of terminating the alkylation reaction, concentrating the proteins for analysis, and removing ionic impurities that interfere with IEF. When cells were lysed in "buffers" that support the activity of enzymes such as lysozyme and benzonase, the conductivity of the resulting lysate was an order of magnitude higher than when lysis was induced by chaotropic urea detergent solutions. Following reduction and alkylation, the conductivity of both lysates was lowered by ultrafiltration to the 0.1-0.2 mS/cm range in preparation for IEF. The detergent 3-(4-heptyl)phenyl 3-hydroxypropyl dimethylammonio propanesulfonate (C7BzO), which favors the solubilization of proteins, but which interferes with SDS equilibration and second dimension PAGE, was effectively removed by ultrafiltration and exchanged with CHAPS without measurable loss of protein. Disparate protein patterns of Rhodopseudomonas palustris lysates were revealed by two-dimensional gel electrophoresis depending on which reagent was used to induce cell lysis.

Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection

“…1) Detection Sensitivity-A dilution series of the recombinant protein mixture was designed to provide data points above and below ϳ1 ng of protein, the accepted detection threshold for SR (6,9,15,19,44,64). The lowest limit of detection (LLD) was defined as the smallest quantity of protein that could be statistically distinguished from background and thus consistently detected (t test, p Ͻ 0.05).…”

Two-Dimensional Gel Electrophoresis Reveals Differential Protein Expression Between Individual Daphnia

“…Centrifugation assisted the ultrafiltration, and the samples were exchanged with fresh UTC until the final DTT concentration was 10 mM. Reduction and alkylation of the samples were performed directly in the ultrafiltration devices using 5 mM tributylphosphine and 50 mM acrylamide as described (Smejkal et al, 2006a). The alkylation reaction was terminated by resuming centrifugation and ultrafiltrative exchange.…”

Tris interference in IEF and 2‐DE

“…The two lysates were combined and centrifuged at 24 000 RCF for 20 min to pellet cellular debris. The supernatant was transferred to an ULTRA-4 centrifugal filtration device in which the alkylation reaction was performed [10]. Tris (40 mM) and 10 mM acrylamide were added and alkylation proceeded for 2 h. The alkylation reaction was terminated by centrifugal exchange into 7 M urea, 2 M thiourea, 4% CHAPS, and 40 mM Tris.…”

“…Following IEF, the IPG strips were equilibrated for 2610 min in SDS equilibration mixture containing 105 mM SDS, 3 M urea, 0.01% phenol red, and buffered to pH 7.0 with 50 mM Trisacetate. (Lower urea concentration has been prescribed for SDS equilibration of IPGs [10], since at higher urea concentrations, decreasing the molar fraction of water alters the micellar properties of SDS, thus affecting the rate of equilibration.) Second-dimensional PAGE was performed in 113 mM Tris-acetate polyacrylamide gels (14061006 1 mm using 50 mM Tris, 50 mM Tricine, 0.1% SDS running buffer.…”