I've recently experienced some problems in denaturing SDS gels. The sample was boiled and run on a 3% stacking; 10% resolving gel. I obtained some bands in the gel as normal, but in some wells I obtained bands right at the top of the resolving. I concentrated the samples and the band tends to increase in intensity. Mass spec confirms the presence of my proteins. Is it possible that it is an aggregation that impossible to break down and hence cannot move in? The proteins have been through size exclusion and ion exchange and then loaded on the gel. The concentrated samples are shown here.
Hi there,
Some agregates may resist to SDS/PAGE sample pretreatment (heat combined to reductive condition and presence of SDS). If you want to solubilize these I would suggest to add 8M urea to your sample buffer.
Have you tried adding more sample buffer with SDS to your protein solution. Also, try adding fresh reducing agent to make sure you are breaking the disulfide bonds if any. If you still have problems, then try the 8M urea method suggested above. If this doesn't work then it may also be likely that you have a high molecular weight impurity in your sample that survived your previous cleanup procedures.
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