Hello everyone. I need some help troubleshooting my SDS-PAGE. My marker seems to not be resolving fully with the last band coming out being 40kDa. I have attached a picture as an example.
My running conditions are:
10% separating gel and 4% stacking gel, both hand cast
150V for 1 hour 30 min
using the Bio Rad Mini Protean system
Any input would be greatly appreciated!
Well, since that band according to you corresponds to 40 kDa, it seems likely at least for me, that the running time is excessive and that the other bands of the molecular weight marker and part of the protein sample went out of the gel. But that is weird because the dye front is visible right at the bottom of the gel. However, if you look at the lane next to the marker you can see a band of protein in the edge of the bottom of the gel, so the protein might be getting out.
What molecular weight marker are you using? Is it pre-stained? If the marker is pre-stained I recommend you during the run, to be aware of the smaller marker and once you notice it is at the bottom of the gel, you stop the run
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