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Protein inhibitor(s) during immunoprecipitation ?

T. Chyau Liang tliang at utmmg.med.uth.tmc.edu
Tue Mar 19 11:41:48 EST 1996

In article <TI6MxNAugZTxEw3F at alexisuk.demon.co.uk>, "Dr. Stephen Hoare"
<s.hoare at alexisuk.demon.co.uk> wrote:


> PMSF (phenylmethylsulfonylfluoride) is not a user-friendly reagent
> (highly toxic). AEBSF (4-(2-Aminoethyl)-benzenesulfonylfluoride,
> hydrochloride) also known as Pefabloc SC is a safer alternative which
> can replace PMSF in most applications. It is used at the same
> concentration as PMSF. Another big advantage of AEBSF is that it is
> water-soluble and stable so that stock solutions can be prepared.
> MW 203.23 . 36.46    C8H10FNO2S . HCl    CAS = 30827-99-7
> It is available from Alexis Corporation (cat. no. 270-022).
> Dr. Stephen Hoare
> Alexis Corporation (UK) Ltd.

Dr. Hoare brings up an important point, i.e., PMSF is not stable.  Its
half time in pH 7.5 buffer is about 5-10 min. Therefore, it should never
be added in the buffer until right before use. Also, one manufacturer
suggests that you make a stock solution in isopropanol. This is not a good
advice, since PMSF reacts with alcohol. Although isopropanol, due to its
steric hindrance, reacts with PMSF slower than other alcohols, you are
better off with DMF, DMSO, or dioxane. You can freeze this stock solution
and use it for long time. I don't have data on AEBSF, but I will still
make its stock solution in organic solution rather than adding it to the
stock buffer or alcohol stock solution. 

I will add some general discussion of proteases, which are classified into
four classes: serine, cysteine, acid (aspartic), and metallo proteases.
The following are inhibitors specific for each class:
  *serine:  PMSF (broad specificity), aprotinin (specific for trypsin-like E).
  *cysteine: NEM, PCMB (p-chloromercurylbenzoic acid), E-64 (a natural
product with an epoxy functional group that reacts with cysteine).
  *Acid: pepstatin
  *metallo: EDTA, phenanthroline, phosphoramidon.

BMB has good literature information on most of this.

Hope this help.

T. Chyau Liang, Ph.D.
U. Texas-Houston

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