Problem with Isoelectric Focusing on gels

Tom Anderson univ0938 at herald.ox.ac.uk
Fri Jul 4 09:24:27 EST 2003


On 4 Jul 2003, Christian Collin-Hansen wrote:

> Question: Is it nessecary to fix IEF-gels in TCA after focusing (prior
> to staining)? Are there alternative ways of fixing the gel?

firstly, how about just using more TCA? i used to use 20% for protein
precipitation.

secondly, i would hazard a guess that IEF gels can be fixed like any other
polyacrylamide gels, in which case you might try some combination of
alcohol and acid; 40% ethanol, 10% acetic acid has worked for me.

alternatively, if you really can't fix the protein, blot it, then stain
the membrane. although if you've got those plastic-backed strips, that
might not be an option.

the only other thing that springs to mind is that it might be a staining
artefact; maybe your protein is there, but due to its evidently unusual
nature, isn't detected by silver stain. you might try coomassie or sypro
ruby (or similar) instead. this is probably a rubbish suggestion.

anyway, IANARSY, but there you go.

tom

ps 'real scientist yet' 8)

-- 
skills to pay the bills!






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