I am a new PhD. student and it is my first time working with PVDF membrane but I have worked with nitrocellulose successfully.how can I preserve my PVDF over the weekend and how can I develop the PVDF membrane to obtain best results as I already worked with one and got loads of background noise?
Honestly, you'll get the best results if you carry out the rest of the western blot without trying to preserve it over the weekend. We tend to work on weekends (welcome to graduate school).
Hi Joshua,
There are two things you need to keep in mind while working with PVDF membranes. 1) These membrane need to be activated by soaking in 100% Methanol prior to use for blotting proteins from the gel. 2) After blotting, one usually needs to block the membrane with 5% skimmed milk or 5% BSA (both prepared TBST) and this is true for nitrocellulose membranes as well. You need to make sure that your membrane NEVER gets dried after blocking as this will definitely give you a lot of background.
As far as the weekend story goes, you can always leave the blot in TBST and leave it at 4 degrees both before and after blocking and then continue the process on the next working day. I have worked with PVDF for a long time and this usually works just fine.
Hope this helps. Good luck!
Utkarsh
Hi Joshua,
High background is usually associated with the specificity of your primary antibodies, and to a lesser extent the strength of your washing buffer and how vigorous you wash your membrane. I have worked with PVDF a lot and as Utkarsh mentioned, do not let your membranes dry. I sometimes have to leave my membranes over the weekend as well, and what I usually do is wrap them in Saran wrap and box them. Membranes stored this way can stay good for a week.
Best of luck.
Rachel
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