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Transfer basic protein to PVDF - (Jan/05/2010 )

I will transfer a basic protein to PVDF. The pI of the basic protein is ~11. I used Tris-Glycine transfer buffer with 20% methanol. Can I adjust pH of this transfer buffer from 8.3 to 12 with NaOH?


no. that pH is out of the effective buffering range of the solution. you may be able to use a phosphate (pK 12.3) or piperidine (pK 11.1) buffer.

caps has an effective buffer range of pH 9.7-11.1, you can try a caps or caps-phosphate buffer.

by the way, pI is not as important if you are running a sds gel (the sds will impart a net negative charge to the protein over a broad pH range). if not sds then you may be able to reverse the electrodes or switch sides with the membrane and use the standard tris-glycine-methanol buffer.

also, if not using sds, then methanol is not required in the buffer (if using a pvdf membrane you will still need to activate in 100% methanol).