I have been doing western blot for the last 8 years using Immobilon P (Millipore Sigma cat #IPVH09120). Since there were zero issues with the transfer, I never paid attention to what grade of MeOH we were using. In my new lab where I oversee every purchase, I bought a laboratory-grade MeOH from Fisher (#Cat A411-4) and found that the western transfer had severely compromised (observed through faint PS marker bands and almost no ponceau S stain) using the same old protocol (500 mA, 1 hr transfer) and even the same apparatus, pvdf membrane, etc. I never thought that the MeOH could be an issue as it was mentioned nowhere in the PVDF manual. After chasing so many permutations of possible causes, I found that only the MeOH is an issue. I called my previous lab and found that we used Sequencing grade MeOH free of peroxide (Fisher #Cat BP1105-4). And when I switched to this methanol, the transfer issue was resolved. I just thought of sharing this observation with the folks here if they are facing such a situation. Thanks.
Hello Anirban Kundu ,
very interesting !
Do you think there is something specific about your westen blot protocol that could explain why you need sequencing grade MeOH while I assume most lab and myself don't have a problem when using "regular" MeOH ?
Cheers,
Philippe.
There might be an issue with this lot of Immobilon P membrane...cannot confirm.
Best ponceau staining on PVDF membrane can be achieved by presoaking the transferred membrane (1 min) in 100% MeOH before adding ponceau.
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