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
At times, it happens that upon stripping protein bands are not at all visible in the membranes. This is because the stripping buffer may change the pH of the membrane and hence prevent the antibodies from binding to the protein in the membrane. Do not worry, repeat the WB once again with the new antibodies. I hope you will get the results.
Yao Xinyi Le Chang Anupriya Bandyopadhyay Thank you all for the kind answers!
I did once again and it worked really good!! But I changed not only the TBS-T, but also the transference configuration and I used the antibodies already diluted. :)
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