I'm performing a western blot with samples that I need first to treat with a solution (yellow) to derivatize carbonyl groupS (the assay is called Oxyblot). My protein samples (bone protein extract) are in 1X SDS-PAGE loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS,6% Glycerol, 1% β-mercaptolethanol and 0.004% bromophenol blue) and when I add the derivatizing solution it turns yellow. When then I run the samples, in the same gel the Molecular Marker and a control, not treated with the derivatizing solution, run fine but the samples that are yellow run very slow and at the end I see a big staining at the start point of the gel with some few bands. Now I find out that BMP is a Ph indicator and when it turns yellow means the ph is acid. The question is: do acid samples have problem running in a sds page gel? What can I do?
If your protein solution turns yellow it means the proteins in the buffer mixture is too acidic. But the protein solution will obvious become blue in 1x running buffer pH 8.3 to pH8.8 range if your not following standard SDS-PAGE. As for molecular marker you better try Precision Plus ProteinTM WesternCTM Standards (Bio-Rad). I belief its the best marker out there for both Western blotting and standard SDS-PAGE. I hope it helps
Hi, Marilina. You can try to neutralize them by sucking up some ammonia
vapour with a pasteur pipette and blowing it into the sample (sample buffer already added). You should see them turning back to blue
Hi, I am also having the same problem but when I did coomassie staining for the same sample they run fine and the bands also look fine which means samples do not form aggregates, it is just western blot or detection problem. When I do the western blot and detect them as mentioned in the protocol (S710, Oxyblot protein oxidation detection kit) I see big stain at the start of the gel. I am not sure whether all my oxidized proteins are of higher molecular weight. Would like to know what you think.
I added a little of Tris 1.9 M pH 8.8 in order to obtain the bleu color in the samples. If the sample is yellow is acid and doesn't migrate well in the gel
Marilina Piemontese I was just wondering if you ever managed to solve this problem as I am experiencing the same difficulties now using the OxyBlot kit. In the end, did you alter the pH of your samples to get them to run better?
the samples run better. I don't alter the pH of my samples. In this way I adjusted the pH because if the bromofhenol blu becames yellow it means that the solution of the sample is acid.
By adding 2 uL of 1N NaOH to my samples prior to loading on my SDS-PAGE I have now managed to get the samples to run nicely and to actually be able to detect them on my blot. That worked for me. Just wish Merck had put something about the pH in their protocol. Would have saved a lot of time.
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