Dear Arvind,

Here is a homemade cocktail, designed since my times back as a post-doc in the US in the 1980s (no kit, no commercial cocktails at that time):

PIM x 100 (PIM = Proteases Inhibitor Mix)

For 25 ml EtOH 25% in water (final volume)

2.5 ml of Aprotinin at 2 mg/ml (in water)

1.25 mg of Leupeptin

2.50 mg of Pepstatin A

39.2 mg of Benzamidine (MW 156.55)

47.5 mg of Sodium metabisulfite (MW 190.1)

PMSF 100mM

It's a x100 solution so you dilute it 100 times.

Best wishes

Prof Philippe Urban

It depends on the composition of protease inhibitor which you are using. Sometime it is a mixture of leupeptin, pepstatin and PMSF and if it is not then you can add. What generally I do, I add these protease inhibitors including PMSF separately.

Dear Arvind,

Here is a homemade cocktail, designed since my times back as a post-doc in the US in the 1980s (no kit, no commercial cocktails at that time):

PIM x 100 (PIM = Proteases Inhibitor Mix)

For 25 ml EtOH 25% in water (final volume)

2.5 ml of Aprotinin at 2 mg/ml (in water)

1.25 mg of Leupeptin

2.50 mg of Pepstatin A

39.2 mg of Benzamidine (MW 156.55)

47.5 mg of Sodium metabisulfite (MW 190.1)

PMSF 100mM

It's a x100 solution so you dilute it 100 times.

Best wishes

Prof Philippe Urban

Since, it has all the component including PMSF, so I think no need to add  extra PMSF 

Thank you to both of you.  The Protease inhibitor Cocktail I was supposed to use consists of the following components

AEBSF-HCl --- Serine protease inhibitor

Aprotinin--- Serine protease inhibitor

Bestatin --- Amino-peptidases inhibitor

E-64 --- Cysteine protease inhibitor

Leupeptin --- Serine and Cysteine proteases inhibitor

Pepstatin A --- Aspartic acid proteases

Still I need to add PMSF. Apart from being a protease inhibitor those it have any other function?

Hi Tarana, AEBSF is a more stable version of PMSF and does the same job, so no, you do not have to add PMSF.

PMSF is very unstable in water, so its really better to add it immediately before or after lysis. Like AEBSF, it is a suicide inhibitor, so once it inactivates a protease, that protease is gone for good. If your samples are very sensitive to proteases, there may be some small value in adding PMSF to a cocktail already containing AEBSF, as it may have nonoverlapping functionality (this is pure speculation, as we don't fully understand all proteases in yeast!)

HI,

AEBSF is a stable alternative to PMSF. So, I presume, that there is no need to add PMSF.

Hello, 

You do not likely need to add PMSF, unless you are particularly worried about serine protease action and want to spike the mixture with extra insurance. 

Thank you everybody for your valuable comments.

Hey Justin. Just a small comment on you saying that PMSF (or AEBSF) is a suicide inhibitor. From my understanding of suicide inhibitors (substrate-like compounds that form an irreversibly and covalently bound intermediate and thus  inhibit the enzyme) PMSF isn´t one. PMSF just binds to the catalytic serine and thereby inhibits the enzyme. Or do I get sth wrong?

I agree with all above answers .