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Problems with signal in WB

Problems with signal in WB - Western Blot Forum

Problems with signal in WB - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 02-15-2011, 10:52 PM
Pipette Filler
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Unhappy Problems with signal in WB



Hi,
I have been doing wb since I started the phd and they always were ok, but now I'm having problems with a new set of samples. I thought the problem was the amount of protein because I could not see any protein, even the actin, but I repeated the western and the signal was right. Yesterday I did a stripping of the membrane which has also workd always and I didn't get any signal. It is not that I don't have signal, I have a weak signal or in one case I had no signal.
I have done the tbstween 1x and the milk tha same day as I did the western and the antybodies are recycled so they work.

Could you help me?
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Old 02-16-2011, 12:04 AM
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Default Re: Problems with signal in WB

Hi there could be your developing steps - is the developer working fine?
What about the chemiluminescent solutions? Are they old? Are other people's western's working ok?
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Old 02-17-2011, 05:12 PM
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Default Re: Problems with signal in WB

The steps after the ponceau are ok because it looks good and the chemiluminiscence solutions are the same as the others use and their westerns are ok. I think it could be related with the removal of the ponceau, that maybe is not totally correct.
Somebody has advised me to do it with the transfer buffer and today my western was really good!
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