I did this week but i got some problems. I run the gel to detect endothelial NOS (t eNOS and p eNOS) in different mice genotype (HT, WT and AMPKalpha1 KO), I used anti t eNOS and ant p eNOS as a primary antibodies and Donkey anti mouse green and Donkey anti rabbit red as secondary anti body, and the DSD gel was 15%. The band was not clear I do not know what is the matter.
I am trying to compere between t eNOS and p eNOS in different mice genotype
Any one can help me to solve this matter?
Good morning!
In my experience, when my Western bands are not clear, its because there´re some isoforms in the extract. I think that, maybe, you are using a high concentration of acrilamide, so you can try to use 12,5% (is not to much difference but your bands will be more separate). You can also try changing the incubations times with your primary antibody.
Good luck!
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