4 replies to this topic

[mefromparadise]

Hi everyone,

I am putting together a protease inhibitor cocktail for my protein. I figured I need PMSF, EDTA and benzamidine in it at the same time to keep it from degradation. The problems I've been having is:

1- Can I premake and mix all the above reagents in some solutions? I thought this was a bad idea since PMSF doesn't dissolve in water whereas benzamidine and EDTA do. Also I'm not sure how these would react in high concentrations with each other.

2- If I make a 100mM PMSF/Ethanol solution, wouldn't that ethanol in there denature my protein? Assuming that I'm adding it directly to the protein I elute off the Ni-NTA column?

3- How often do I need to add protease inhibitors? I was told that PMSF acts covalently (hence I don't have to add it IF i add it to the lysis buffer) but doesn't PMSF degrade quickly? therefore it will lose its bonding properties?

Any inputs on how to inhibit proteases at the max would be appreciated

[Vini]

mefromparadise, on Feb 13 2010, 02:34 AM, said:

Hi everyone,

I am putting together a protease inhibitor cocktail for my protein. I figured I need PMSF, EDTA and benzamidine in it at the same time to keep it from degradation. The problems I've been having is:

1- Can I premake and mix all the above reagents in some solutions? I thought this was a bad idea since PMSF doesn't dissolve in water whereas benzamidine and EDTA do. Also I'm not sure how these would react in high concentrations with each other.

2- If I make a 100mM PMSF/Ethanol solution, wouldn't that ethanol in there denature my protein? Assuming that I'm adding it directly to the protein I elute off the Ni-NTA column?

3- How often do I need to add protease inhibitors? I was told that PMSF acts covalently (hence I don't have to add it IF i add it to the lysis buffer) but doesn't PMSF degrade quickly? therefore it will lose its bonding properties?

Any inputs on how to inhibit proteases at the max would be appreciated

Hi, I dunno if this wud help u. But, i make my protease inhibitor in the following way and it works:
1.5mM Benzamidine, 2mg/L pepstatin (stock made in DMF) and 2mg/L leupeptin. This is the working concentraion that I use in disruption buffer and make it from separately prepared stock solutions for each.

Best.

[mdfenko]

mefromparadise, on Feb 12 2010, 03:04 PM, said:

3- How often do I need to add protease inhibitors? I was told that PMSF acts covalently (hence I don't have to add it IF i add it to the lysis buffer) but doesn't PMSF degrade quickly? therefore it will lose its bonding properties?

free pmsf will decompose rapidly in an aqueous environment. the bound pmsf will not be affected (it is already altered: serine protease + pmsf → irreversible enzyme-pms complex + hydrogen fluoride).

make up the pmsf stock in anhydrous alcohol (we most often use methanol, sometimes isopropanol). you should add fresh to each solution immediately prior to use.

[mefromparadise]

Thanks! VERY helpful!

Quote

Hi, I dunno if this wud help u. But, i make my protease inhibitor in the following way and it works:
1.5mM Benzamidine, 2mg/L pepstatin (stock made in DMF) and 2mg/L leupeptin. This is the working concentraion that I use in disruption buffer and make it from separately prepared stock solutions for each.

Best.

Made this and it worked! I added 10mM EDTA too!

Quote

free pmsf will decompose rapidly in an aqueous environment. the bound pmsf will not be affected (it is already altered: serine protease + pmsf → irreversible enzyme-pms complex + hydrogen fluoride).

make up the pmsf stock in anhydrous alcohol (we most often use methanol, sometimes isopropanol). you should add fresh to each solution immediately prior to use.

I figured this out the hard way! I kept adding protease inhibitor to my purified protein every day as I was concentrating it down and by the time it was down to the volume I wanted it in, I had close to zero protein! I just found out WHY! LOL

[mdfenko]

mefromparadise, on Feb 24 2010, 12:17 PM, said:

I kept adding protease inhibitor to my purified protein every day as I was concentrating it down and by the time it was down to the volume I wanted it in, I had close to zero protein! I just found out WHY! LOL

how did you concentrate? if you used a membrane, are you sure it wasn't damaged? had an exclusion limit low enough to retain your protein? did you flush the membrane to remove any protein that settled and stuck to the membrane?