I routinely perform cell fractionation and subsequent protein localization studies with Escherichia coli. All protocols I have come across used EDTA as chelating agent to avoid proteolysis during cell fractionation. Now I plan to purify my proteins from independent fractionation samples and I predict EDTA present in the buffer may ruin the whole purification experiment (I use a His-tag and nickel-based purification protocol). How necessary is to include EDTA during cell fractionation? Is it enough to use additional protease inhibitors (cocktails, PMSF …) to avoid proteolysis?
I would say not required at all. I rarely use any form of protease inhibition prior to protein purification. My default is to keep additions to purification buffers to an absolute minimum and then only add things like EDTA or an inhibitor cocktail if it appears to be absolutely necessary. Even for proteins which appear to be showing proteolysis I generally find I can solve this by keeping the lysis/fractionation timing to a minimum and purifying at 4C.
Thanks for your reply. As you claim, it should be fine as long as everything is done at 4ºC. I will include no EDTA in my future experiments, thanks again for your opinion.
If you are purifying a recombinant protein expressed in a strain of E. coli usually recommended for heterologous expression, there is no need for inhibiting proteases, these strain were manipulate to express very low amount of proteases. In any other occasion I used a commercial cocktail of inhibitors just in the extraction buffer avoiding it during purification.
It all depends upon your lysis procedure. The main reason that EDTA is present in many cell fractionation protocols is to remove the Mg that stabilizes the outer membrane.
So if you get good lysis without EDTA then you should be ok, but if your lysis is not working efficiently, then this may be the answer. it all depends upon the protocol.
@ Tozzi: I do indeed express my proteins in a E. coli strain with low protease expression.
@ Benedik: I have tested my lysis efficiency including no EDTA and aparently I get decent cell lysis and reasonable protein yields after fractionation.
I am performing my experiments excluding EDTA and I do not notice any difference comparing to previous results. Of course, everything done at 4ºC and/or ice. Thank you both for your advice.
Hi there. Using protease inhibitors in your fractionation experiment is more than necessary. EDTA addition is suggested in many protocols bacause of its potential to inhibit metalloproteases, given the reaction Mg^2+ + EDTA^4- → MgEDTA^2-. However, in our lab we do not add extra EDTA in the subcell. extraction reagents as we wash our cells previously in Buffer containing EDTA 2mM, which does the job satisfactorily never causing any problems with purification.
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