My hunch is that your sample is to blame. You should run your previously positive sample alongside to ensure your POI is indeed present in your new samples. Good luck!

Hi Kurtis: I always advise probing for your POI first and then the control afterwards to confirm loading etc. Your POI maybe much less abundant than your control B-Actin, which appears to have been "burned in" to your membrane as it was not removed by your stripping buffer. Next time you run B-Actin with these samples I would dilute the antibody significantly.

So you may not be seeing your POI because the control is so evident you are not exposing long enough to see your POI because the control would be very overexposed. Also, be sure your expected band sizes are not to close to each other as the very bright B-Actin could obscure the POI band.

As far as the temperature you are using to denature, if it was too high you would see evidence of smearing or streaking in your lanes so if that is not present then the Temperature is most likely not the problem.

Hope this helps.

Bethany McGonnigal thank you! I will try my POI first and dilute my B-actin control. Hopefully that works.

If you recycle your antibodies, the recycled antibodies may contain other previously used antibodies that are released from the blot.