Hello.
I prepared protein lysates from Hek293t cells by using RIPA buffer. When I run my samples on a SDS gel, they don't separate. I have attached pictures of the gel after running it and of a comassie staining. Does anybody have an idea what the problem here could be?
What is the protein size and gel percentage?
Follow the attached protocol and you have to load 20 ug of protein and the total volume 15-18 ul per well.
Good luck
Hi Alissa,
It looks like too much protein or that the protein is not completely denatured.
Do you clear your samples by centrifugation before adding the loading buffer (i.e. laemmli buffer)?
What's the formulation of your loading buffer? Did you boil your samples followed by centrifugation before loading them in the gel?
Cheers,
Sebastián Dupraz I centrifuge the samples to clear them and boiled them and centrifuged them before loading. I use a commercial sample buffer from Alfa Aesar. The formulation is: 375mM Tris-HCl (pH 6.8), 9% SDS, 50% glycerol, 9% beta-mercaptoethanol, 0.03% bromophenol blue. It's 6
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