I stained a western blot gel with coomassie dye that has been used multiple times before. The staining worked, but it was very light. First we thought that the protein concentration in the sample was too low, but we measured it and its at 0,815mg/ml. So this shouldn't be the problem.
Is it possible that the dye wasn't working well anymore.
PS. I noticed some (blue) sediment in the dye before using it.
The dye is consumed by the processing of dying the gel. Reusing the stain solution will eventually deplete the dye from it. In addition, the dye becomes contaminated by SDS from the gels, and diluted by the liquid content of the gels. Fortunately, the poorly stained gel can be stained again with a fresh stain solution,.
Reusing the stain after the washing with buffer? Maybe this is the case. Has it been used many times? (I would say 3 times as maximum)
Mª Angeles Zorrilla Lopez-Perea No, I put the gel into the staining solution right after the PAGE. But as Adam B Shapiro said, I think the answer is to simply stain it for longer, or using a fresh staining solution.
Use can reuse many times. Stain 10 minutes add destain heat in microwave
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