I am having some issues with the use of GuHCl during gel electrophoresis, I use GuHCl to solubilize Ab peptides before loading them in SPE, and of course in the flowthrough collections I have a huge amount of this chaotropic agent. Basically, when I boil the sample before loading in the sample buffer, the fractions containing 4M GuHCl forms granular material that is really awkward to load. I already tried to load the sample without boiling but I always observe a "smiling" effect during the run, sometimes also the loss of the peptides in the FT fraction.
How can I prevent this ?
I have no experience with your specific method, but with general denaturing His pulldowns we had similar problems.
One possibility (the expensive option) is to use a purification/concn kit like the pierce one. http://www.piercenet.com/product/sds-page-sample-prep-kit
The cheaper option will be to substitute GmCl with Urea if possible and avoid boiling before loading. Hope it helps.
The kit seems really good, because is quoting exactly my kind of issue! I will try to convince my supervisor to buy it, and meanwhile I can try with Urea or some spin cup step to eliminate GuHCl! thanks for the suggestions
Manoj's suggestion is a good one. You could try micro-spin desalting columns with G25 , G50 sephadex - depends on the size of the peptide. The smiling effect is due to high salt. You will still get some smiling with urea though not so much pptn. If you give your boiled peptide-GuHCl-SDS mix for 5min spin in a microfuge and only load the "soluble fraction" is it any better ? (you will probably still get some "smiling" since you will not have removed the GuHCl.)
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