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Ponceau red problem: membrane turned red


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#1 yulia24

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Posted 14 March 2013 - 11:34 AM

Hi everybody

I've done Ponceau staining of my PVDF membrane after the transfer and got a lot of background binding that doesn't go away after washing with water. My whole membrane is red, with transferred bands visible on that red background. Does it mean there is no point continuing with this membrane? Anyone can explain why on earth did it happen?

Thanks

Y.

#2 bob1

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Posted 14 March 2013 - 11:40 AM

Did you block it before adding the ponceau? If so, this is the problem - the block causes protein to be all over the membrane.

Try washing the membrane in a protein containing solution (e.g. 3% non-fat milk powder in TBS-t).

#3 yulia24

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Posted 14 March 2013 - 11:41 AM

Nope, stained straight from the transfer.

#4 GNANA

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Posted 14 March 2013 - 11:58 AM

i guess some pH issue, i heard in low pH destaining is hard with ponseau.....continue washing with TBST, it suppose to vanish...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....




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