i needed to check cell signalling pathway, after treatment cells with some Conditioned media at some time curse . lysed and collected samples as usual. did bardfoard protein assay, samples had good amount of proteins. then ran samples on 8% gel. transfered the gel on PVDF membrane . after transfer i stained my samples with ponceau. nothing. marker got transfered but not my protein sample. i had another gel running in parallel.. and i could see ponceau staining there.i blot my samples with antibody membrane was empty. can't find any explanation.
Hi Amani,
Ponceau doesn't work very well on PVDF membranes. You could use other stains (i.e. amido black) to make sure that your proteins are there.
Hope this helps.
Thank you guys , yea i believe so too, as further i was able to see my protein of interest on membrane during detection.
thanks for the advice ajit will try to do this method for the sake of curiosity :)
The same happend to me and I was afraid of having transferred for too long (wet tank western blot transfer, 2 hours, RT, 200 mA). Did you try with amido black? Did it work?
hey anna. no I haven't . but lately i am doing the transfer using nitrocellulose and not pvdf, and even skipping the ponceau staining
Is it needed to wash PVDF membrane in methanol for ponceau stain because methanol already added in transfer buffer ?
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