Semi-dry western blot transfer procedure

Semi-dry western blot transfer procedure:

This protocol describes the procedure for transfering protein from
polyacrylamide gel onto nylone membrane using semi-dry transfer
method.

1. Cut 6-10 sheets of whatman 3MM the same size as the mini PAGE gel.
2. Cut membrane (nylone or immobilon-p PVDF) to the size of the gel.
3. Membrane activation: Wet PVDF membrane in methanol for 15 seconds
(don't need this step for nylone membrane) and then transfer to a
container filled with ddH2O for 5 min. Soak membrane in 1x transfer
buffer for 10 min while preparing transfer sandwich.
4. Soak whatman filter paper in transfer buffer for several minutes to
10 min to avoid trapping of air bubble.
7. Assemble sandwich: (Bottom up on the surface of wet semi-transfer plate)
a. 3-5 sheets of wet whatman filter paper
b. membrane
c. polyacrylamide gel
d. 3-5 sheets of wet whatman filter paper
8. Use a roller to roll over the sandwich gently to remove trapped air
bubbles. Apply few ml 1x transfer buffer on top of the sandwich to
avoid drying out during membrane transferation.
9. Transfer at 14 voltage for 40-60 min.

Note: It is recommended to transfer very large proteins by wet
transfer other than semi-dry transfer.

1x transfer buffer recipe:

25 mM Tris
250 mM Glycine
15% methanol

Last update 29-Aug-2006, Rating Very Good of 2 votes.