I ran SDS-PAGE with 14% lower gel, 4 % upper gel, having brushing, pre-running empty gel for 30 min.
The protein samples had been centrifuged with 14,000g for 10 min, and then the supernatants were added with 5X commercial sample buffer heating 100 °C for 10 min. After cooling and spin down, the samples were instantly loaded to the wells.
But with many tests, the samples cannot run down, shown as the figure.
Can anyone help me? thanks a lot!!
Try running blue native PAGE in case your proteins form aggregates.
Instead of boiling the samples, heat them in sample buffer at 70°C for 15mins and reduce the resolving gel percentage to 10-12%.
Hope this solves your problem.
Yuting Chiang does your commercially available sample buffer come with beta-mercaptoethanol and if not, did you add any?
Beta-mercaptoethanol is required to break disulphide bonds and fully unfold proteins so that they migrate properly.
Yuting Chiang I am not sure whether its coz of protein aggregation, probably your sample may contain high molecular weight proteins.
You can reduce the SDS PAGE % to 10 or 12, just give it a try.
Also you can try with DTT based loading dye instead of Beta-mercaptoethanol based one so that you can avoid heating of your samples.
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