Preparative polyacrylamide gel electrophoresis

Abstract

We have studied several variables affecting the migration and resolution of protein mixtures in preparative polyacrylamide gel electrophoresis. In the system we have used (Uniphor column, L.K.B.) the maximum sample load is 10 mg of protein per cm 2 per protein band. When separating two closely adjacent bands, this value must sometimes be lowered to as little as 2 mg of protein per cm 2 per protein band. Of all the bed gel heights tested, the range 5–10 cm seems to be the most useful; shorter gels are convenient only when the components to be separated differ markedly in size or isoelectric points; longer gels have the disadvantage of increased electrophoretic times and consequent diffusion of the protein bands in the gel. For most of the protein species usually found in biochemical analysis, gel strengths ranging from 4 to 8 % acrylamide are most suitable. In particular, when dealing with a heterogeneous sample, a 4 % gel is convenient as it allows all the protein species to enter the gel. As for the elution buffer rate, we suggest the range 12–25 ml/h; slower rates will not remove the sample from the elution stopper efficiently, with the consequent risk of re-mixing the separated bands, while higher rates will bring about a marked dilution of the sample applied. It is advisable to have in the gel and in the electrode chambers a buffer of low ionic strength (20–30 m M); this allows a high potential gradient to be applied across the gel, thus shortening considerably the electrophoretic run. Sample recoveries in the system used are all greater than 90 %.