I need to transfer two very basic proteins (pI of 11.4 and 11.04) and would like to know what transfer buffer is most suitable for these proteins when run on a denaturing gel. CAPS buffer has the highest pH that I am aware of, but at pH 11 I think that it's still not basic enough for the transfer of my proteins. The predicted molecular masses for these proteins are 22 kDa and 33 kDa, respectively so I worry that adding too much SDS in order to increase the negative charge of the protein will cause it to transfer through the membrane.
What transfer conditions do people working with basic proteins (> pH 11.4) under denaturing conditions use?
Would simply placing the membrane on the cathode side of the gel while using a Tris-glycine transfer buffer be an option?
And lastly, would using a 0.7% acetic acid transfer buffer (with the membrane on the cathode side of the gel) be an option even though it's a denaturing gel?
Seems you are able to resolve your proteins in SDS-PAGE. So they also should transfer with "normal" buffers. If your afraid it might travel in the opposite direction, just put another membrane there. Or use passive transfer by sucking the buffer from the gel into filter paper on both sides and put blotting membranes between.
Have you ever tried the Dunn carbonate transfer buffer? It is also suggested (pI >9) in denaturing gels even at higher pI like 11. It can also enhance the ability of some antibodies to recognize and bind to antigenic sites on proteins. CAPS is more efficient at the transfers of larger proteins rather than small ones like 20-30kda.
Since you choose nitrocellulose membrane to transfer you can try to eliminate or decrease the amount of methanol at Dunn or CAPS transfer buffer. Alcohol may cause a reduction in pore size, precipitation of some protein, and some basic proteins to become positively charged or neutral. All of these factors will affect blotting efficiency.
In SDS-PAGE (unlike native) the charge depends on the bound detergent, not the amino acid composition of the protein.
I second Ismail's recommendation of Dunn's buffer, I have found it both cheaper and better than Towbin's.
You may also try CTAB-PAGE for separation, it may at least give you a better estimate of the molecular mass.
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