Samples are important, so I decided to go forward with my experiment. The Running buffer was used without SDS. Can I save my experiment t?
Hi Ali,
I am afraid, without SDS in ur gel the technique is no longer SDS_PAGE... its more like a native gel with a SDS_PAGE sample buffer... that is the precise reason for all the wierd observations.. just repeat the exp with proper gel preparation and i guess u will be fine with it!!!
Good luck
Ali, you don't have much choice, probably, so simply go ahead. I'm afraid that you might end up with unsharp or even smeared bands, however.
That's why we are calling it re-search...
Hi Ali,
I am afraid, without SDS in ur gel the technique is no longer SDS_PAGE... its more like a native gel with a SDS_PAGE sample buffer... that is the precise reason for all the wierd observations.. just repeat the exp with proper gel preparation and i guess u will be fine with it!!!
Good luck
Your sample and your gels contained SDS, I presume. Thus, at the beginning of the electrophoresis, proteins will run normally. However, as SDS moves faster than proteins, there will be depletion during the run, and the charge of the proteins, and hence their velocity, will drop. This will affect the Rf-values. Since all lanes of the gel are affected in the same way, you should be able to compare the results within the gel, but not between this gel and those run normally.
At any rate, I'd incubate the gel in warm running buffer for, say, 10-15 min to allow SDS to bind to the proteins again. Otherwise, transfer may not work for lack of charge. Only then switch to transfer buffer and proceed normally.
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