PMSF? - (Sep/09/2008 )

HI just wondering can anyone tell me - I added PMSF to my RIPA buffer when I lysed my cells however now I am equalzing them (and Im using RIPA buffer again...) do I need to add the PMSF again to the RIPA buffer just before use of can I use RIPA buffer with out PMSF?

Thanks

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you will dilute PMSF in the second case, but you will have added in excess anyway;

in the future, try to become independent from PMSF for several reasons...

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I also don't know the answer to this question, so I'm buzzing it to keep it on top.

do we need to re-add PMSF or other protease inhibitors prior to normalizing samples again?

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We don't use PMSF in RIPA buffer. We use ortho-vanadate, pNpp and Protein Inhibitor Cocktail (Boehringer).

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PMSF has a very short half life, once it is in contact with water. The half life is only about 20 minutes. Therefore, always prepare your RIPA buffer with PMSF fresh.

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Personally I think using the protease inhibitor cocktail would be a better choice to target different types of proteases present after cell lysis. PMSF has short half-life and need to be dissolved in anhydrous ethanol or anhydrous isopropanol. So I end up did't use it...I plan to add in protease inhibitor cocktail which has better half-life and it's easier to dissolve (just need to dissolve in buffer or distilled water or can directly add into the lysis buffer).

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Great advice, PMSF should be made up fresh, just before adding it to the lysis/protease cocktail that you use.

Rhombus

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My understanding is that PMSF permanently inhibits serine proteases, so you shouldn't have to add more. I don't know about the other protease inhibitors

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that's what I thought too, there is no need to re-add PMSF.

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PMSF is very unstable in the
presence of water. The half-life of aqueous PMSF at
25°C at pH 7.0, 7.5, and 8.0 is 110, 55, and 35 minutes,
respectively.

---From SigmaAldrich product information page.

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