Hello fellow researchers,

I'm having a puzzling problem in my Western blot experiment, can somebody help me? I conducted an experiment using prefrontal cortex samples, following a WB protocol that has previously yielded successful results (We took quite some time to standardize each step). However, this time around, I'm facing a situation where I'm not able to detect any bands, despite thoroughly checking various aspects of my protocol.

Here are some key details:

- My samples were homogeneized in RIPA buffer + proteases inhibitors as usual, and are relatively fresh, I homogenezeid last month, and I am realizing western blot with those samples since that.

- I run my electrophoresis in BioRad system, at 150V, 400mA, 2h, room temperature (10% acrylamide gel)

- I transfered to nitrocelulose membranes in semy dry transfer 30V, 1h, 164mA, room temperature

- I performed a Ponceau staining and confirmed that the samples were transferred correctly to the membrane (image is attached)

- I used three different antibodies in those membranes in the first time (I cut the membranes in three different sizes), it didn't work and I thought that it could be a old antibody solution problem. So I stripped the membranes and I incubated with new antibodies solutions (I got three new and sealed antibodies, including the secondaries) and none of them resulted in detectable bands.

- I was very careful to see that I incubated the correct primary antibodies, with their respective secondary antibodies

- Blocking (BSA 5%) and washing steps (3x with TBS-T) have been successful in previous experiments with those antibodies of the same brand.

- The protein quantity in the samples appears adequate, as good bands were visible in the Ponceau staining.

- I'm using high-quality and well-maintained Super-ECL reagent.

I'm completely stumped by this situation, especially because even the internal control protein, beta-actin, is not being detected. If anyone has faced a similar issue or has suggestions on what else I can investigate, please share your insights. Any assistance or guidance would be greatly appreciated.

Thank you for your attention and help!

Nicolle Platt