Factors affecting the range of pH gradients in the isoelectric focusing dimennsion of two‐dimensional gel electrophoresis: The effects of reservoir electrolytes and loading procedures

Abstract

We have examined methods for broadening and stabilising pH gradients used in the first dimension of two-dimensional gel electrophoresis. The replacement of typical strong electrolytes with weak electrolytes as reservoir anolyte and catholyte allows the generation of broad (3.5 pH unit) gradients that are stable for at least 28 000 volt x hours (Vh). Protein patterns form within 10 000 Vh are stable throughout the subsequent focusing. Large quantities ofprotein, up to 6 mg, can be loaded onto such gels by mixing samples into the gel prior to polymerisation. We found no evidence for extensive modification of proteins because of exposure to the polymerisation reaction. The pH gradient formed in strong electrolytes in the typical O'Farrell gel system (J. Bid. Chem. 1975,250,4007-4021) could also be broadened ifthe samples were again mixed into the gel before polymerisation. This last effect appears to be due to an effect of the sample buffer used upon end loading of samples. J. A. A. Chambers et al.

Solubilization of Proteins in 2DE: An Outline

“…This induces a stacking of the proteins at the sample-gel boundary, which results in very high concentration of proteins at the application point. worked with nonreduced samples [27,28]. Although this presence of disulfide bridges is not optimal, inclusion of the sample within the gel has proven of great but neglected interest [27,28].…”

“…worked with nonreduced samples [27,28]. Although this presence of disulfide bridges is not optimal, inclusion of the sample within the gel has proven of great but neglected interest [27,28]. It must however be pointed out that it is now possible to carry out The problem is however more severe for hydrophobic proteins when an IPG is used.…”

Two‐dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte‐pH gradients

“…This could be reduced but not abolished by protecting the sample from the acidic electrolyte by a liquid layer containing 20% glycerol and carrier ampholytes, but penetration of the proteins remained poor, probably because of some coprecipitation with the acidic proteins unable to enter the gel. We therefore decided to use weaker acid as electrolytes and especially Good's buffers, since their use has been reported to favor pH gradient stability [20,21].…”

Solubilization of Proteins in 2-D Electrophoresis: An Outline

“…Owing to this situation, most workers describing inclusion of the sample within the IEF gel have worked with nonreduced samples [27,28]. Although this presence of disulfide bridges is not optimal, inclusion of the sample within the gel has proven of great but neglected interest [27,28]. It must however be pointed out that it is now possible to carry out acrylamide polymerization in an environment where disulfide bridges are reduced.…”

“…iodoacetamide, N-ethyl maleimide) also inhibit acrylamide polymerization. Owing to this situation, most workers describing inclusion of the sample within the IEF gel have worked with nonreduced samples [27,28]. Although this presence of disulfide bridges is not optimal, inclusion of the sample within the gel has proven of great but neglected interest [27,28].…”