I'm having trouble to detect phospho-ACC2 in a western blot from white adipose tissue samples. I have some nice blots, but in the last few weeks I can't repeat the process. Anybody know how to improve the detection?
Hi Daniel, we had similar problems in skeletal muscle. We were able to solve it by using a different tissue lysis method. The key is to preserve the phopshorylation as it is not very stable. It is better to use fresh lysate and if it is not possible to keep your lysates in -70 or liquid nitrogen. For lysis we use just laemmli sample buffer, briefly, we homogenize the sample on ice in X1 laemmli sample buffer with phosphatase inhibitors and boil it right away, to preserve the phosphorylation. Next we spin down the samples and run the supernatant.
Hope it helps.
Good luck.
- Badges
- Science method