Rapid staining of proteins in ultrathin‐layer isoelectric focusing in polyacrylamide gels

Abstract

AbstractA method for protein staining after isoelectric focusing (IEF) in polyacrylamide gel is described in which the total time for fixation, staining and complete gel background destaining is reduced to 3–5 min. This is achieved by (i) staining dry gels on silanized supports instead of hydrated gels, (ii) reducing gel thickness to 50 μm, (iii) using Servalyt carrier ampholytes with good destaining properties, and (iv) using dyes with low affinity for carrier ampholytes, namely Serva Violet 17 or 49, facilitating destaining of gel background. The sensitivity of staining is 100–200 ng protein/ mm2 over a 10 cm separation distance and 20–30 ng protein/ mm2 over 3 cm gels (miniature focusing). Addition of 5–20 % methyl acetate to the staining or destaining solution reduces the sensitivity of staining significantly. Most effective fixation of proteins in 50 μm gels is achieved with 20% trichloroacetic acid. In solutions of alcohol and acetic acid, appreciable amounts of proteins are leached after incubation for 5–20 min. Paraformaldehyde, malondialdehyde or glutaraldehyde (5–10 %) exhibit no fixative effect on proteins in 50 μm gels when used directly or after precipitation of the proteins with trichloroacetic acid. The selective leaching of proteins under a variety of experimental conditions shows that adequate fixation is particularly important in work with basic, low molecular weight and/or hydrophobic proteins.