Add the protease inhibitors immediately before starting the homogenization process. If your target is susceptible to degradation by endogenous proteases, it will be degraded quite rapidly once they proteases are released by homogenization of the cells. Frozen samples are less prone to degradation by proteases, so I would suggest add the inhibitors at the beginning and try to work on ice or in a cold room as much as possible.

Add the protease inhibitors immediately before starting the homogenization process. If your target is susceptible to degradation by endogenous proteases, it will be degraded quite rapidly once they proteases are released by homogenization of the cells. Frozen samples are less prone to degradation by proteases, so I would suggest add the inhibitors at the beginning and try to work on ice or in a cold room as much as possible.

Hi there,

Add them to the resuspension/lysis buffer.

Some lysis and thus release of proteases is always occurring so you want to minimize that process. We too add protease inhibitors to our resuspension buffer, so before we aliquot and freeze the cells. If we work with a very prosease-sensitive protein we also add inhibitor (in an amount proportional to the aliquot of cells) when we thaw the cells, as my predecessor suggested.

Thanks for everyone's answers- seems like the consensus is at the point of homogenisation, and working on ice.  regards, S

Hi Stephanie, the previous answers are correct. Just remember that you should always protect the proteins at all stages of the process.  Not sure why you are worried about the presence of EDTA/EGTA in buffers as it will not be present after transfer to the membrane for blotting.  I would cover my bases and use the widest array of inhibitors that you can.

Also, the presence of EDTA and EGTA may reduce activity of metalloproteases, by chelating divalent cations as Ca+2

HNS

I would add them freshly just BEFORE homogenisation.

Tissue out and into ice cold lysis buffer (with inhibitors already present in buffer) asap.  Keep temperatures down during homogenisation by doing short bursts and chill on ice in between.  If using a sonicator add some ice to the water.  Keep your frozen samples aliquotted to reduce freeze thaw cycles.  If you want to make the aliquotts close to single use you might want to keep the samples on ice and quantify protein immediately so you know what a sensible volume is for aliquotting.

As suggested by Maximillian, you have to add antiproteases (APs) before homogeneisation/lysis. However, you have to take in account some additionnal points:

1/ some APs like PMSF are not really stable in water (t1/2 ~30'). So you have to add it only just before lysis (some minutes) to be sure it will have any effect.

2/ Action of APs on proteases is quite slow. If some proteolysis happened, you can probably try to do lysis at 4°C or to increase the concentration of APs.

As others mentioned, its best to add protease inhibitor (better use protease inhibitor cocktail, that inhibits different proteases activity) before homogenization. I would also recommend you to add protease inhibitors before and after each step once you ruputre the plasma membrane  i.e after homogenisation, after lysis and solubilisation.