This is my first post on the forum; I appreciate any help I can find here. Thank you so much!

Our lab is exploring the role of the AMPK / ACC pathway in fatty liver disease. We drug-treated cells from MEF and AML12 cell lines and generated samples to run in Western Blot.

Our problem is that for P-AMPK and P-ACC, we are getting really high background and the lanes look streaked from top to bottom, many times to the point where we can't see the banding clearly. For ACC and P-ACC, the streaks are especially dark up to the first protein marker (example photos attached).

My team suggested that the samples may be too concentrated or some proteins do not enter the gel well at high MW. We hence tried to dilute the sample or to load half of what we usually load in our protocol, and tried Novex Tris-Acetate gels once. We also tried to use a weaker chemiluminescent substrate at lower exposure times and it helped somewhat.

Here's a summary of our protocol:

- samples are prepared w/ 4x loading buffer and both BME and DTT master mix prior to loading

- using precast Novex Tris-Glycine wedgewell gels

- both electrophoresis and transfer are done with a BioRad system: Run gel at 135V at the default 2 amps setting for 1 hour, and transfer to PVDF membrane at 20V for 1 hour with transfer buffer containing 10% methanol

- we block the membranes with BSA

- currently we are using AMPK/ACC antibodies from Cell Signaling at 1:1000 dilution, and secondaries at 1:2000