I am characterizing different snake venoms. As snake venom is a mixture of various large protein complexes (with both acidic and basic proteins) i want to run native agarose gel electrophoresis with lanes in the middle of the gel bed. My question is what is the ideal running buffer for this? can i use regular TAE buffer?
Thank you in advance!
Sreyasi.
doi:10.1016/0003-2697(82)90113-0 has recipes for electrophoresis buffers of different pH. TAE-buffer is probably less suitable, as Tris and acetate have very different pKa-values.
The choice of buffer pH is determined mostly by the pKi-value of the protein, at a pH below pKi the protein has a negative charge and moves toward the positive electrode (kathode). Above the pKi proteins are positively charged and move towards the negative electrode (anode). If pH = pKi, the protein is uncharged and does not move at all.
These considerations apply to native electrophoresis, in the absence of reducing agents and detergents. In the presence of detergents, charge is determined mostly by bound detergent molecules, not by the amino acids of the protein.
One other aspect: The paper mentioned above uses continuous electrophoresis. If you want to make use of the concentrating effect of discontinuous (DISC) electrophoresis to get crisper bands, you need not one, but two buffer systems as explained in doi 10.1111/j.1749-6632.1964.tb14207.x :, these buffers are carefully matched. doi:10.1111/j.1749-6632.1973.tb47551.x gives recipes for that situation.
Thank you Engelbert for your help!
I will definitely go through the paper. Actually, i don't know the individual pK values of venom components (as it is crude venom), and also I don't have a gradient maker so can not run gradient electrophoresis. So i thought about native agarose gel electrophoresis.
You need to use a buffer which is alkaline, say tris glycine or similar so that the protein if interest has a net negative charge and hence moves through the gel towards anode.
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