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Transfer basic protein to PVDF


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#1 yueh76

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Posted 04 January 2010 - 10:30 PM

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?

#2 mdfenko

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Posted 05 January 2010 - 11:31 AM

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).
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