Has anyone tried loading additional protein onto a Western blot after it has started its run? I'm in a position of having some very dilute samples and needing to load a large amount of protein (40ug/well). Other than trying to concentrate the sample and the desalting process, etc., I thought maybe I could "double-load" the well - after it has run about 10 minutes and before it is out of the stack, I could try adding additional sample.
Has anyone tried this? Did it work? Am I crazy to think this might work?
Hello Mary Nivison
How's about using a broad comb or a thicker glass plate (1.5-2.0mm) which can accommodate more volume of sample?
I don't think it's crazy. With luck, the stacking gel might stack it into a single band just fine. Why don't you try it, let us know what happens? However when I'm in that situation, I would use acetone precipitation. It's very easy, no desalting needed.
If you don't have wider combs, you could make the concentrator gel a little wider than normal and load more sample as you enter your sample. You could also use concentrators, but you will lose at least 10%, if you use this put 5% glycerol to prevent your protein from sticking. Or the precipitation with acetate that Jonh suggests.
Dear Mary Nivison
In addition to were mentioned above, I find it is practically to use 10x or 8x loading buffer without equalizing by distilled water, consequently you can load higher volume of the sample.
I just did it now, and I will keep you posted when I image for P-FAK and GADPH. I hope so much it works.
Update: it works amazingly perfectly. pic is uploaded. I ran the gel for 2 min only, then reloaded the rest of the samples and continued the exp.
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