Hi Rachana, It may be possible that the protein in your sample may have degraded due to inclusion of some proteases. Do you add protease inhibitors to your protein lysates? Also, I would highly recommend making aliquots of your lysate after quantifications and store them in -80 C

, if possible, to avoid repeated freeze thawing cycles.

Thank you Sanchita for your answer. Yes, i do add protease inhibitor and store my lysates at -80 degrees.

Protein degradation will degrade the proteins and i might get series of bands corresponding to the protein! However in my case i get sharp bands (may be not in this attached pic but i have other examples). I am trying to TCA precipitate or heat the samples to 65 degrees. Lets see how that goes!

Hello Rachana L. Patnayak

Have you thought of changing the loading control? In mouse tissue samples, not all loading control proteins are expressed equally. Moreover, under certain experimental conditions, the protein levels of some loading control proteins can be affected. Therefore, it becomes essential to check reliable expression databases and the literature to ensure the tissue type expression of loading control protein before you perform Western Blot. Make sure that a given loading control protein is highly expressed in your tissue sample before running any blots. It may be possible that the loading control is not expressed at constant rate in your samples.

I have attached an article below which may be helpful.

Article Mouse Adipose Tissue Protein Extraction

Best.

Hi Rachana, As Malcolm mentioned that in mouse tissue samples, not all loading control proteins are expressed equally. You may try a different loading control.

Besides, you may try Ponc

eau stained blot and if that shows equal intensity of your loaded protein lysates, that will also prove that you have loaded equal amount of protein for all your samples.

Malcolm Nobre Sanchita Mallick

Thanks for your response. Yes, I have tried beta actin, GAPDH, and hsp90.