I am trying to measure adiponectin expression in 3t3 cells using Enzo's adiponectin antibody. I have been using 1% Milk 1% BSA as my blocking buffer and for all other steps of the western blot; this is not working. I was wondering if anyone else has had success with this, with other solutions. Thank you!
Dear Rebecca,
I am assuming that you have used well differentiated 3T3L1 adipocytes for your experiment. As differentiation step is a must for adiponectin expression.
Secondly, you can optimise the blocking buffer concentration, time and temperature of incubation.
I have not performed western blot for adiponectin but for my protein of interest I have always prepared 5% BSA blocking buffer. Since skimmed milk was not a suitable option for phosphorylated proteins. You can try to keep the membrane for overnight blocking at 4 degrees.
Let me know how your troubleshooting works...
Best luck!
Swati
Like Swati already implied, it is best to compare 3T3-L1 lysates of before and of after induction of differentiation side-by-side. Block overnight and incubate primary for 1h. If problems persist, perhaps the integrity of the antibody needs to be questioned.
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