Bio-Rad’s Colloidal Gold Total Protein Stain is a stabilized gold sol (i.e. suspension) optimized for rapid and sensitive identification of proteins bound to nitrocellulose or PVDF membranes. Staining of proteins bound to nitrocellulose or PVDF membranes can be done by Amido Black, Ponceau or colloidal gold stain. In my experience, maximum sensitivity is achieved using colloidal gold stain. Proteins which are present in very minute amounts generally cannot be visualized by Ponceau or Amido Black stain.
The Colloidal Gold Total Protein Stain comes in a single size: 500 ml. It can be used to stain proteins bound to nitrocellulose or PVDF membranes. The other reagents required for the staining procedure are routinely-used chemicals. Staining can be done even after performing immunodetection on membranes; the only requirement for this is to strip the membrane in destainer containing 40% methanol and 10% acetic acid for 30 minutes. The staining protocol is very quick. After transfer of proteins onto the nitrocellulose or PVDF, membranes are washed for 20 min in TBST (20mM Tris pH 7.5, 500mM NaCl, 0.1% Tween-20) 3 times. After washing, membranes are rinsed with water for 2 min to remove all salts, which might interfere in colloidal gold staining. Membranes are then incubated with colloidal gold stain (50 ml is sufficient for a 15x15 cm membrane) for 1 h; incubation can be increased to improve the staining. After incubation, membranes are washed with water to remove excess stain. The colloidal gold can be reused 2 times depending on the required staining intensity and the quantity of proteins on the membranes.
My research aims to identify proteins/tumor antigens which elicit an antibody response in cancer. It involves separation of tumor cell proteins on 2D gels followed by transfer of proteins from 2D gels to PVDF membranes. In order to detect proteins which elicit an antibody response in cancer patients, these PVDF membranes are immunostained with IgGs purified from sera of cancer patients, followed by chemiluminescent detection with X-ray film. The resulting film image shows reactive spots which represent tumor antigens. To find out which spot on the film corresponds to which spot on the silver stained 2D gel, the film must be overlaid onto the silver stained 2D gel, but there are problems in overlapping images due to differences in the sizes of 2D gels. These differences occur because of the different conditions used during silver staining and the transfer of proteins from 2D gels to PVDF membranes.
This problem was solved with Bio-Rad’s Colloidal Gold Total Protein Stain. Because 2D blots can be stained with colloidal gold even after immunostaining, film and colloidal gold stained 2D blot images can be overlapped. It is then easy to correlate spots from colloidal gold stained 2D blots to the silver stained 2D gels because colloidal gold stains all of the proteins on the 2D blots which can be visualized by silver staining. However, the appearance of some spots on colloidal gold stained 2D blots is different than on the silver stained 2D gels. This difference is a result of the different chemistries involved in the different staining methods. These differences are not great enough to create problems in comparing colloidal gold stained 2D blots with silver stained 2D gels.
Colloidal Gold Total Protein Stain can also be beneficial for staining blots obtained by one dimensional electrophoresis, where small amounts of protein are typically loaded. The band intensity obtained by colloidal gold staining can also be used for quantification.
I would, therefore, recommend Colloidal Gold Total Protein Stain for quick and sensitive detection of proteins on membranes, in spite of the marginally extra cost of colloidal gold.
Sanjeev Shukla
PhD Student
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Biochemistry and Cell Biology Dept