About basic proteins and basic membranes: adjusting blotting and staining conditions to Immobilon CD

Transfer efficiency from polyacrylamide gels and binding to Immobilon P and CD were tested in different buffers with 125I-labelled proteins. With a derivatized poly(vinylidene difluoride) membrane, a pH 8 medium was found to be superior to a more alkaline solution, for both acidic and basic proteins. New staining protocols were tried on Immobilon CD. Toluidine Blue and iodine vapour gave a negative and a positive stain, respectively, with a fair band-to-background contrast. Protein sequencing after both stains was not impaired by interfering peaks. Biuret solution stained the protein bands pale pink but, even after copper removal with a chelating agent, it completely prevented Edman degradation. The first two procedures compare favourably with a commercial kit for protein detection, Quick Stain, that provides comparable sensitivity but results in several spurious peaks on protein sequencing.