Dry or not PVDF membrane after transfer - (Nov/16/2010 )
Hi, i was wondering if it is necessary to dry PVDF membrane AFTER transfer. Millipore suggests to do so, in order to get optimal results (lower background). But when i am going to incubate with BSA or milk for blocking is it a must? And when you dry and the bands become visible, do i have to incubate with PBS-Tween before blocking? I mean i dry the membrane with the idea to hydrate it with PBST? What's the purpose of doing it? Thank u for reading it im looking forward for your responses.
Although this is a suggested step, I never did it and never had any issues with my western blots. Perhaps if you do your blot and have background issues, it would be a good idea for the next time. If you do choose to dry your membranes, you should rehydrate before blocking. The instructions with my PVDF (Millipore, Immobilon-FL) says to re-wet the dry blot in 100% methanol for 15 seconds before placing in blocking. From what I've read, the purpose of drying the membrane is so that the proteins are more adherent to the membrane. This might be especially important if you know you are going to need to strip the membrane to get all your desired blots but again, I've never had any problems with membranes that I didn't dry, even after stripping.
rkay447 on Tue Nov 16 16:32:23 2010 said:
Although this is a suggested step, I never did it and never had any issues with my western blots. Perhaps if you do your blot and have background issues, it would be a good idea for the next time. If you do choose to dry your membranes, you should rehydrate before blocking. The instructions with my PVDF (Millipore, Immobilon-FL) says to re-wet the dry blot in 100% methanol for 15 seconds before placing in blocking. From what I've read, the purpose of drying the membrane is so that the proteins are more adherent to the membrane. This might be especially important if you know you are going to need to strip the membrane to get all your desired blots but again, I've never had any problems with membranes that I didn't dry, even after stripping.