bioprotocols.info - Electroblotting Proteins onto Nitrocellulose Membranes

Electroblotting Proteins onto Nitrocellulose Membranes

Electroblotting proteins onto Hybond-c extra; supported nitrocellulose membrane.
The following protocol is for proteins separated on standard SDS containing polyacrylamide gels.

Soak gel in transfer buffer for at least 10-20 minutes.
Equilibrate membranes by briefly soaking membrane completely in ddwater and then in transfer buffer for at least 10 minutes.
Using a commercial electroblotting unit transfer proteins onto membrane.
Remove membrane and wash with PBS or TBS to remove residual acrylamide.

Transfer Buffer:

Bjerrum and Schafer-Nielsen transfer buffer for SDS-Proteins using nitrocellulose (with methanol) or Zeta-Probe membrane (withoutmethanol) (1).

Recipe:
48 mM Tris, 39 mM glycine, (20% methanol) pH 9.2.
Dissolve 5.82 g Tris and 2.93 g glycine [and 0.0375 g SDS or 3.75 ml of 10% SDS] in ddH2O (add 200 ml of methanol); adjust volume to 1 liter with dd H2O.

DO NOT ADD ACID OR BASE TO ADJUST pH. The buffer will range from pH 9.0 to 9.4, depending on the quality of the Tris, glycine, ddH2O, and methanol. Methanol should be analytical reagent grade, because metallic contaminants in low grade methanol will plate on the electrodes.

Note: Some pH electrodes will not perform a proper measurement for the pH of Tris buffers. If the pH of the buffer is not correct, check the electrode to be sure it is designed to function with Tris buffers. If the pH electrode works properly with Tris buffers. and the pH is below 9.0, remake the buffer.

(1) Bjerrum and Schafer-Nielsen, C., Analytical Electrophoresis, M. J. Dunn, ed., p.315; Verlag Chernie. Weinheim, (1986).