Dear Sonal,

To my knowledge the purified 20S proteasome is only able to degrade proteins that are entirely unfolded; i.e. denatured in an ATP-independent manner. Thus, I'd suggest to treat your BSA and calmodulin as you did with oxidizing agents and before the proteasome degradation assay, you should denature the proteins somehow.

For example, by heating them up (>70°C), adding urea or GdnHCl, adding organic solvents or reducing agents, precipitation with acetone, etc. After denaturing your proteins, if you didn't do it by high temperature, you should remove excess of denaturing agents or diluting your sample before testing the proteasome degradation. Please also check the reported temperatures of denaturation for either protein and if they stay unfolded when you put them back at the assay temperature for your proteasome assay.

You may also want to double-check if your oxidizing agents are effectively removed and not affecting the proteasome. Please make sure to perform proper controls to check if these agents interfere with proteasome degradation. If possible, better to remove the oxidizing agents after treating your proteins by rounds of buffer exchange (e.g. using amicons) or precipitate your proteins with cold acetone, rinse and resuspend in fresh buffer.

Hope these suggestions help, best,

Alfredo

Thanks for your suggestion Alfredo, I am removing oxidizing agents via filtration and dialysis. If I use urea or GdnHCl it will unfold the protein but If I remove it will my protein fold again? How to keep protein unfolded? The paper I am following just used H2O2 for oxidation and nothing else and their experiment is working.

Dear Sonal,

Thanks for the extra details. In general, each protein has its own folding-unfolding features. Not all proteins can fold back to the native state by themselves after denaturation. A lot of proteins need chaperones or assembly factors to get to their native states. You should check the literature to know if your proteins of interest have a reversible folding after treatment with denaturants or high temperature. If they are still unfolded upon removal of denaturants or decreasing again the temperature, then you may have a chance to get proper degradation by the 20S proteasome.

I quickly checked the information from companies, and apparently you can activate the purified 20S proteasome by adding a bit of SDS (0.035%) or another activator called PA28. If this is true for your assay, then you can also think in denaturing your proteins with SDS (e.g. 1%) and then dilute the protein and detergent prior to the proteasome activity assay. The remaining SDS should keep the proteins unfolded, or at least make them less prone to refold.

Good luck with your experiments!

Best,

Alfredo