Hi everyone,
I'm interested to perform a semidry transfer for a Western Blot. Right after activating the PVDF membrane for 30 seconds on pure methanol, do you directly place it on the gel, or do you still do something else? (e.g. wash the membrane with ddH20 or with transfer buffer). And if so, any specific reason?
Thanks in advance!
Dear Enrique,
I follow this protocol:
- wet the PVDF membrane in 100% methanol for 15 seconds until change from opaque to translucent
- place the membrane in Milli-Q water and soak for 2 minutes
- place the membrane in transfer buffer and let equilibrate for at least 5 minutes
-place quikly the membrane on the gel , avoiding it to dry
After activation in pure methanol, and before placing on the gel, wash the PVDF membrane by soaking in transfer buffer solution with gentle rocking.
To activate PVDF membrane, put it in pure Methanol for 2 min. then wash it by using transfer buffer and immediately place it on the gel. if you are not using ladder, do not forget the notches :)
Good luck
As per the others who replyed, after soaking with MetOH (I do for 10 min), carefully rinse wih ddH2O (like 3x5 min or so) then incubate with Transfer buffer.
Good luck!
Soak the PVDF membrane in methanol for 2-3 min and then wash or rinse in water three times. Then soak it in transfer buffer (optional). Directly place it on the gel. Works every time.
Same answer as everyone else, equilibrate the MeOH wetted membrane in transfer buffer for at least 5 minutes. As for why you do this, my understanding is that you want to make sure that there is a continuous ion concentration between the electrodes at the start of the electrophoretic transfer.
PS - This is a very helpful guide from EMD Millipore:
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.fishersci.com/content/dam/fishersci/en_US/documents/programs/scientific/brochures-and-catalogs/guides/emd-millipore-protein-blotting-handbook-guide.pdf&ved=0ahUKEwiCr-G-8dTXAhUE6oMKHT0SAY0QFghAMAQ&usg=AOvVaw2eU3Gke8TEIf3VcbndtlfP
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