Prem, Since I work on a tight budget, I tend to make my own protease inhibitor cocktails. The tablets that are available from BD Scientific, Roche, or Sigma work terrifically well, but they are pricey. Therefore, I buy my own protease inhibitors and then make frozen aliquots of a common stock solution. You need to use a mixture of protease inhibitors, since no one protease inhibitor will inhibit all available proteases. I make a stock solution of 100 mM PMSF that I usually dilute 1/50-1/100. I also use a stock solution of 100 mM benzamidine HCL that I dilute the same. My stock solution of Pepstatin is at 100 µg/ml that I dilute 1/20 and 100 µg/ml stock solution of Leupeptin that I dilute 1/10. With this combination of protease inhibitors and by keeping your extracts ICE COLD, you will usually be able to inhibit any problematic proteases. Oh - EDTA (pH 8) at a final concentration of 1mM is also important for metalloproteases.

@ ms biotech , here there are four components given in the cocktail and concentration of individual component is provided. So should I add all the individual concentration and take it as an stock solution ?

Prem,

Adding protease inhibitors to a cell lysis is an _experiment_, just like any other. The correct answer is that you need enough to prevent the proteolysis that you want to avoid (which PMSF clearly doesn't do), but not too much more to minimise cost.

I would try doing several small scale trials with a small amount of your cell pellet, and varying amounts of the inhibitor cocktail (at that price, I'd be hoping that the cocktail would last 2-5 L worth of culture at least). Work out how much you need to add to get proteolysis down to an acceptable level, and then scale up.

Hi Prem,

I found this on the german Merck Millipore site:

Good luck,

Magdalena

Hi Magdalena

First of all I would like to thank you for replying my question. I read this data sheet but no information about the amount to be added is being provided.

Well Prem, look for the stock conc. that calbiochem is providing. Always use working conc. as 1X.

What tag is on your protein? We purify His6 tagged proteins and use 1mM PMSF, 1mM benzamidine HCL, 7ug/ml Pepstatin, 5-10µg/ml Leupeptin and 10 µg/ml Aprotinin or Protease inhibitor tablets from Roche diagnostics (complete mini, EDTA free - 1 tablet per 10 ml lysis buffer). As far as the reagent you are using, it comes lyophilized and should be reconstituted in1ml DMSO (should be provided with pkg) with 4ml dH2O then aliquoted and frozen (stable up to 1 month @ -20 once reconstituted). At this concentration i've used it at 40ul per 500ul buffer (you will need to vortex right before use because it will tend not to go into solution). This link will provide you with the concentrations of proteases in the reconstituted solution, therefor, you can add the appropriate amount based on your purification protocol (if you are performing a bradford assay, yuo will need to check the max amount of each that can be present)

http://wolfson.huji.ac.il/purification/PDF/Protease_Inhibitors/CALBIOCHEM-Inhibitors.pdf

Hi Norah,

My protein has an affinity tag complementary to the structure of NADP and I purify it through a dye affinity column. No His tag is present in it. I have seen the data sheet and here after reconstituting it the individual components has their individual final concentration in 5ml solution. On the basis of which protease inhibitor stock concentration I should make the working concentration before cell lysis? Do you have any suggestion for this ?

I use a protease inhibitor cocktail from BD (Baculogold) made for insect cells, the pepstatin A is at 0.7 mM in the 50X stock solution, so your stock solution is around 100X in regards to pepstain A. But then you need to decide how many cells (either based on OD 595, or weight of cell pellet) you lyse per mL of lysis buffer with the inhibitors at 1X, that is very important. I would contact the Calbiochem tech support to see what they recommend. Remember to keep everything on "wet ice" when lysing the cells.

Hi, usually I make a 50 X stock solution of a commercially available cocktail and add 1X to my lysate volume i.e 1X (of the stock 50X) to my final volume of lysate. The lysate needs different conc of individual inhibitors, so this way it is not cumbersome and i do not need a lot of calculation just before the exp.

1X to final concentration if its been provided as concentrated form (eg Sigma 100X). When they provide tablets they will tell the volume in which you should dissolve. Eg Roche. These two I have used .

As suggested by Norah, use of complete from roche is a good alternative. Pay attention on presence/absence of EDTA in your cocktail- depending on your experiment, may influence the results.

Prem, Since I work on a tight budget, I tend to make my own protease inhibitor cocktails. The tablets that are available from BD Scientific, Roche, or Sigma work terrifically well, but they are pricey. Therefore, I buy my own protease inhibitors and then make frozen aliquots of a common stock solution. You need to use a mixture of protease inhibitors, since no one protease inhibitor will inhibit all available proteases. I make a stock solution of 100 mM PMSF that I usually dilute 1/50-1/100. I also use a stock solution of 100 mM benzamidine HCL that I dilute the same. My stock solution of Pepstatin is at 100 µg/ml that I dilute 1/20 and 100 µg/ml stock solution of Leupeptin that I dilute 1/10. With this combination of protease inhibitors and by keeping your extracts ICE COLD, you will usually be able to inhibit any problematic proteases. Oh - EDTA (pH 8) at a final concentration of 1mM is also important for metalloproteases.

Many E.coli expression strains have the major protease deleted from the genome. If I suspect protease related problem I add one table from the Roche protease inhibitor cocktail to the pellet from 6-8L culture.

Here is the information you are looking for.

http://www.merckmillipore.com/INTL/en/product/Protease-Inhibitor-Cocktail-Set-II---Calbiochem,EMD_BIO-539132?ReferrerURL=https%3A%2F%2Fwww.google.co.il%2F&bd=1#anchor_BRO