The protease inhibitors will also prevent degradation of the samples during the long focusing steps. Phosphatase inhibitors are also commonly used to prevent dephosphorylation in the event you are looking at post-translational modifications.

Thanks dear Carlos for your response

My mean from this question is about inhibition of protease effects not comparing this inhibitors gel by gel.

Cheers

Dear Jason

What is your idea about using of liquid nitrogen and working on ice during sample preparation?

There is no need of Protease inhibitor in sample preparation for 2D, because you are already using very hoarse conditions like ~8M Urea, 2M ThioUrea, around 200mM DTT, SDS etc. In this situation proteases will also get denatured, therefore they can not affect your proteins.

Thanks dear Santosh

I used tris buffer for creating hemogenate without any of denaturing agents and immidiately store the supenatent at -20 C. At focusing time, rehydration buffer that consist of all of these reagents was used. My 2DE pattern is good but I want to know that are there any differences in 2DE pattern IF I used rehydration buffer in 1st step of sample preparation?

I'm looking for your reply

Cheers

The need for protease inhibitors depends on the sample. When we worked on Cryptococcus (or any other yeast for that matter), as soon as you try and solubilise the protein/break open the cells, the proteases tear everything apart and you end up with a smear at the bottom of a 2D gel. This is in spite of the 7M urea, 2M thiourea, 1% surfactant (C7BzO) present or the reduction/alkylation. Some proteases are really tough (LysC for example, active in 8M urea). Our JPR paper shows this graphically. Bacteria on the other hand don't need inhibitors in most cases.

I use protease inhibitor in human sample. But do not use PMSF because it may modify enzyme. Be careful at working temperature, some protease work well at 4°C (like pepsine). Some others work even in high concentration of urea. Your focusing is done at 20°C. And last, the solubility of urea is not good at 4°C...

to avoid multiple bands due to cleavage and hence false over expressed bands