Hello everyone,
I conducted a native gel electrophoresis experiment for my protein, which has a pI of around 8.5. I used the protocol provided in this link:
http://www.assay-protocol.com/molecular-biology/electrophoresis/native-page.html
I also used a 15% stacking gel. However, I encountered an issue where my ladder stuck, and I can't see any band for my protein.
I'm wondering if the problem is related to the pH conditions or the gel percentage.
Thanks,
Dear Kimia,
The problem seems to be the concentration of acrylamide you used. You're mentioning that the stacking gel was 15%! This is too much, you should use a stacking gel concentration between 3-4%... and the separating gel preferably as a gradient, for example from 4-12%. If you don't have a gradient mixer or the possibility to buy these gradient gels. You should use a concentration that is compatible with the size of your protein. I would go for an 8 or max. 10% acrylamide (continuous) gel. You can check the following reference for a better protocol: Wittig I, et al., Nat Protoc, 2006, 1(1):418-428.
In addition, your protein has a high pI, so make sure that it is correctly charged (negatively) with Coomassie dye, otherwise you may have to invert the polarity to avoid losing your protein in the cathode buffer instead of entering the gel towards the anode.
Best of luck,
Alfredo
Alfredo Cabrera-Orefice
Dear Dr. Cabrera,
Thank you very much for your great help!
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