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Why proteins are not visible on my membrane after ponceau staining?
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+ Biochemistry
+ Proteins
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Larry Isaacs
Why proteins are not visible on my membrane after ponceau staining?
From what you say, I can only give a few advices:
For the quantification, make sure your sample does not contain any substances which disturb the assay. The Bradford assay is for example sensitive against SDS or DTT. See here for more details. This prevents you from loading a sample without enough protein.
Make sure that you really have protein on your gel. Run a gel as normal and then stain it with Coomassie Blue (it cannot be used for western afterwards).
Don't loose your sample - this happens easier than most people think.
Make sure your transfer works. This can either be done by using pre-stained ladders (which are helpful anyway) or by doing Ponceau Red staining. This can be washed off with water and doesn't disturb downstream applications.
Depending on the type of membrane you are using, they might need pre-treatment. PVDF membranes are extremely hydrophobic and need to be activated in methanol.
For the quantification, make sure your sample does not contain any substances which disturb the assay. The Bradford assay is for example sensitive against SDS or DTT. See here for more details. This prevents you from loading a sample without enough protein.
Make sure that you really have protein on your gel. Run a gel as normal and then stain it with Coomassie Blue (it cannot be used for western afterwards).
Don't loose your sample - this happens easier than most people think.
Make sure your transfer works. This can either be done by using pre-stained ladders (which are helpful anyway) or by doing Ponceau Red staining. This can be washed off with water and doesn't disturb downstream applications.
Depending on the type of membrane you are using, they might need pre-treatment. PVDF membranes are extremely hydrophobic and need to be activated in methanol.
I don't see any bands here, but only a smear. I would first suspect degradation of your sample. It would be really important to simply run a gel and stain it to see how this looks like and to find out the problematic step here. It's probably already the gel...More
From what you say, I can only give a few advices:
From what you say, I can only give a few advices:
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