PVDF staining problem... - (Sep/22/2005 )
Hi,
We have some problems in detecting protein on PVDF.
We have stained it with Comassie (it was our first timewith a western blot!!!) 
now tha PVDF after some washing still remains blue and don't appear any signal...
Have you ever coloured a PVDF with comassie?
Do you have any suggestion about it?
many thanks!!!
I was looking into staining my PVDF with a protein dye and came to the conclusion that coomassie is non-reversible ie. you cannot destain it. This is particularly so since use of isopropanol etc results in the coagulation of the protein on the membrane.
Hence, if you need to detect the protein bands and then go on chemi the membrane. you should be using ponceau red. This stain is reversible. However, you got to apply it after the transfer...immediately before the blocking step. Ponceau can be easily destained. I tried the ponceau and it works.
We have some problems in detecting protein on PVDF.
We have stained it with Comassie (it was our first timewith a western blot!!!)
now tha PVDF after some washing still remains blue and don't appear any signal...
Have you ever coloured a PVDF with comassie?
Do you have any suggestion about it?
many thanks!!!
I have stained a PVDF with comassie, but it is very hard to destain, the best thing to do is either stain the gel with comassie or the PVDF with amido black, and destain with water.
Hope this helps.
I'd stain with ponceau red as well. It doesn't interfere with your immunodetection steps either I think so you don't have to destain. Are you trying to check your transfer efficiency? You could coomasie stain the gel to see whats left after the transfer and the transfer of the pre-stained molecular markers is a good indicator.
Ceri