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Western Transfer 1. Prepare transfer buffer just prior to transfer set up. Mix well before use.
 

Summary: 44
Western Transfer
1. Prepare transfer buffer just prior to transfer set up. Mix well before use.
1 Minigel 1 Large Gel
Methanol (ACS) 20 ml 50 ml
ddH2O (4C, from cold room) 70 ml 175 ml
10X BS-N Transfer Buffer 10 ml 25 ml
2. Cut 2 pieces of THICK Whatman paper, about 1 cm bigger than gel. Cut 2 pieces of THIN (3MM)
Whatman paper, about 1 cm bigger than gel.
3. Cut 0.45 m BioRad nitrocellulose slightly bigger than gel. Cut off, or mark, lower right corner of
membrane. Handle nitrocellulose with washed gloves.
4. Slowly submerge thick and thin Whatman paper in Transfer buffer in plastic tray. Do the same
with the nitrocellulose membrane. Allow nitrocellulose to wet for 1-10 minutes.
5. Drain buffer from Whatman sheets and nitrocellulose membrane for a few seconds. Assemble
bottom of transfer stack as diagrammed below.
Remove trapped air bubbles by gently rolling Pasteur pipette across top of stack 2-3 times.
6. Put some transfer buffer on top of the membrane. Dip gel in transfer buffer and place gel FACE UP
onto membrane. Place gel on membrane correctly the first time, if possible.
Remove trapped air bubbles by gently rolling Pasteur pipette across top of stack 2-3 times.
7. Place the remaining Whatman sheets on top. Stack should be moist, but no excess liquid should be

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine