Protein staining on membranes becomes critical when we are working with a system which may not give us equal loading with reference to an internal control in Western blotting. For example, it has been observed that beta-actin expression is altered even in cancer tissues. In such a situation, it is very difficult to order another antibody, standardize and detect again on the same blot. This consumes time, money and effort. Instead, one can use Bio-Rad’s Colloidal Gold Total Protein Stain (Cat No. 170-6527) which is a stabilized colloidal gold solution for rapid and sensitive detection of proteins bound to nitrocellulose or PVDF membrane. Bio-Rad’s Colloidal Gold Total Protein Stain comes in a single pack of 500 ml and is costly as compared to other stains, like Ponceau or fast green which are reversible and Coomassie which is relatively non-reversible.
After performing Western blotting, the membrane should be made free of antibodies bound to the antigen by stripping (2% SDS, 100mM â-mercaptoethanol, 160mM Tris HCl pH 6.8 ) by adding destainer (10% Glacial acetic acid 45% methanol) for 30 minutes. The membrane is then washed with T-TBS followed by 4 washes of 10 minutes with deionized water which is critical to remove any contaminating ions which will interfere with staining by colloidal gold. The membrane is then immersed into a vessel or sealed in a plastic bag containing the colloidal gold stain. The length of the incubation will vary depending on the protein amount. I incubate the membrane for 1 hour for 10 ug of protein loaded/well on a minigel. For 2DGE blots, the incubations vary from 30 minutes to 1½ hours.
One disadvantage is that after staining the membrane with colloidal gold, one cannot use for the membrane for detecting again with some other antibody; unlike Ponceau where the blot can be destained by washing with distilled water. The gold stain is indelible and stays forever. Also, at times there is background. Even when background is present, I still obtain a sharp band of protein, but the background can be enough to make the blot look dirty. To avoid this background, I wash the gel in distilled water several times as mentioned in the protocol; however, some background still remains. Nevertheless, when I dry the same blot (air dry) the background becomes negligible (sometimes disappears). Hence, it has become the protocol of our lab to dry the blot every time after colloidal gold staining. This reduces the background significantly and makes the gel appear more presentable. Also, care should be taken while handling the blot as even the slightest mis-handling of the membrane (like pressing on or bending the blot with forceps) will generate markings on the blot after drying.
The colloidal gold stain is reusable until the intensity goes away. We have used it twice and it works; I have not tried third time. The size of our membrane is 7 X 8 cm and we use 20 ml colloidal gold in a sealed plastic bag.
My lab works on keratins which are intermediate filament proteins; altered expression has been observed during and after malignant transformation. We enrich keratins by treating the biological material (tissue/cell line) with high salt and detergent so as to solubilize all soluble protein. Keratins are insoluble and after centrifugation, the pellet is collected. After protein estimation, we perform 2DGE of these keratins. Now because of enriched keratin, we load 4-8 ug of keratins which gives us good separation. Even the slightest difference in protein amounts are highlighted in the Western blot and also because of the difference in transfer efficiency (if one keeps the blot in different cassettes during electro blotting). Hence, to normalize, we use colloidal gold staining which helps in better analysis of our results. Also, in cancer tissues, the internal controls do not remain controls anymore and for normalization, one has to use a sensitive stain. Analysis is made easy by using colloidal gold stain on the membrane as the sensitivity is close to silver stain. The best part is that colloidal gold staining is quantitative. I would recommend this costly solution for labs working on cancer biology for better analyses.
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Deepak Kanojia
Senior Research Fellow
Department of Biochemistry and Cell Biology
Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
India
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