Dear Colleagues,
After running Western blot on PVDF membrane and detection using ECL, I would like to stain my PVDF with colloidal gold to be able to allign the ECL image with total proteins on PVDF.
Since PVDF has been blocked with skim milk, does colloidal gold bind to the blocking proteins on PVDF?
Do I need to strip the PVDF membrane before staining it with colloidal gold?
Also, I would like to have the most efficient colloidal gold staining protocol that can be used on PVDF after ECL.
I want to get opinions about my concern above.
Thank you
I know that colloidal gold may sometimes interfere with antibody binding if done first, but I do not think you would need to strip the membrane if you already got your antibody to bind and imaged it... I'd try doing it without stripping just to see how that turns out! Good luck, keep us posted!
Thank you indeed, Paul Hafen
Do you have an efficient colloidal gold staining protocol that can be used on PVDF after ECL?
We don't actually do colloidal gold stains to quantify total protein from our blots... we usually do stain-free imaging or ponceau stains for total protein.
But... I think a standard colloidal gold protocol would still work after ECL imaging:
4 x 5 min wash (on rocker)
3 x 2 min rinse with DI water (on rocker) - to remove salts
60-120 min incubation in colloidal gold solution (on rocker) - you can look for bands to determine optimal incubation times
3 x 1 min rinse with DI water (on rocker)
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