Is it possible to run protein samples on agarose gel only?My predicted size of desired proteins is around 30-60 kDa. How much the concentration of agarose should be used for separating protein samples based on predicted molecular weight? I think if I prepare 3-5% agarose gel, it probably can separate protein samples at that predicted MW. All my desired proteins have predicted pI below than pH 7. So, is it possible to run in basic buffer since agarose mixed with 1X TAE buffer and also the same buffer used as running buffer during electrophoresis? Any idea?
What is your problem in running a polyacrylamide gel with SDS? For proteins in your expected range, a 12.5% gel will give excellent results.
Does 12.5% gel refer to separating gel? If I use 8-well comb with 1.0 mm plate, how much do I need to add the volume of protein sample and the recommended concentration of protein sample?
Proteins are folded when they are translated by the cells internal machinery, thus the addition of SDS in the PAGE linearizes the protein before it is separated by electrophoresis. For separating yes, 12.5 is good and a stacking gel of maybe 8-10% would be advisable. Volume for an unskilled hand is usually advisable at 20ul but for careful administration, it is possible to push up to 30ul without spills to other well.
Should be fine I guess, have heard of people using that concentration range but only when their resolving is low to begin with
I would recommend 12.5% separating, 5% stacking. If the proteins being separated are close to the 30k range, 8-10% stacking may not be advisable. The whole idea of stacking is to compact the total protein for better resolution in the separating gel, hence 'separating gel-like' stacking gels should be avoided. If you are using small format gels, like 5x7 cm separating, 20 ul of a good concentration sample is more than enough, remember that's 16 ul protein and 4 ul of the loading buffer. The concentration/amount of protein per well/lane would also depend upon downstream applications, if any, and/or on the detection method to be used: coomassie blue or silver stain.
Well, it is all up to you to trial and error. Different conditions will give different results. If not sure, just stick to the basic protocols supplied by the manufacturer. That is usually a good place to start
If the protein bands can not be seen after staining with coomassie blue and destaining for overnight, should I use silver stain as an alternative way to stain protein band clearly?
You can, but I suggest you first run your gel, do a Coomassie stain and then start to think depending on the result you get.
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