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.

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

GHIBAUDI, Elena Maria;
1995

Abstract

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.
A698
351
359
electrophoresis; blotting; protein separation
GIANAZZA E.; COARI P.; LOVATI M.R.; MANZONI C.; E. GHIBAUDI; SALMONA M.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/5676
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