I have transferred gel in PVDF membrane and stained with ponceau but I couldn't see any bands on the membrane, instead, the whole PVDF membrane is turned into pink color and the color is not going after prolonged wash also. I have used 30 microgram of protein and I transferred in semidry transfer apparatus. I have used the nitrocellulose membrane before that for the same concentration of protein and I could see the bands after stained with ponceau. I got this problem only with PVDF membrane. I have also activated the PVDF with methanol after transfer and stained with ponceau even if I couldn't find any bands. What might be the reason and how to see the bands and how to remove the background pink color?
activate PVDF first before going for transfer. Incubate PVDF membrane in methanol such that whole membrane is submerged for 2-3 min. Then keep membrane in transfer buffer for 2-3 min such that whole membrane is submerged. Not your transer buffer should have 20% methanol plus 0.1% SDS. keep transfer time for 60-80min with constant current of 75mA
activate PVDF first before going for transfer. Incubate PVDF membrane in methanol such that whole membrane is submerged for 2-3 min. Then keep membrane in transfer buffer for 2-3 min such that whole membrane is submerged. Not your transer buffer should have 20% methanol plus 0.1% SDS. keep transfer time for 60-80min with constant current of 75mA
i used to activate PVDF membrane with %100 methanol for like 30sec to 1 min. After that i incubate them in the transfer buffer(%15 MetOH) atleast 5 to 10 mins. as you mentioned, i also use semidry. I incubate pack/paper and the gel itself in the transfer buffer too. when it comes to transfer, i use 25V 2.5A and transfer time depends on your PVDF membrane there are differences between 0,45 and 0,25. You should be able to see bands after this protocol as i saw it before. You may want to stain your gel with commassie blue to make sure the gel itself does not contain more protein than it should have. I also saw people who stained their gel with commassie blue and washed with destain buffer. And they were able to make a retransfer from stained gel and got actual results from it.
And your second q. Most of the people will advise you to wash your membrane with tbs, pbs or tbs-t to remove pink background. I actually advice you to wash it with pure water ( ddH2O ). It will clear the background less than a minute.
After this protocol if you wont be able to see any bands in your membrane ;
- Your transfer time and V/A may cause problems with your membrane.
- Your ponceau red ( make sure you add the acid fresh )
- Your mambrane did not activated (?)
-in some conditions, depends on your transfer time you mambrane can dry out and your proteins cannot be able to transfer to the membrane (semidry).
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