I am looking for a ready-to-use, broad-spectrum protease and phosphatase inhibitor cocktail in order to study the phosphorylation status of specific proteins from whole cell extracts by western blot analysis.
I have used products from Roche, Sigma and currently HALT from thermo fisher. In general any combination of a complete protease inhibitor tablet and phosphatase inhibitor cocktail should work with you big name phosphoproteins (erk, Akt, p38 ikb). Either of these work fine. We make up 10 ml lysis buffer containing protease and phosphatase inhibitors at at time. we can generally keep it from 2 weeks to a month at 4oC with no notable fall-off. Main difference is roche is in a tablet and thermo comes in a liquid form. FYI, I have done similar assays with no phosphatase inhibitors at all, which is acceptable if you run your gels soon after isolating lysates. Hope this helps someone
we use phos stop from roche. Crush one tablet in 10 mL of lysis buffer of choice and you are ready to go. It works very well.
I would also recommend the Roche product for protease inhibitors. It's called complete protease inhibitor cocktail. You can prepare a 50x stock aliquoted in -20°C which you dilute into the lysis buffer (or any other buffer) fresh before use. It combines well with the phos-stop mentioned above as well.
I like Halt from pierce it is a x100 solution, you can get the aliquoted in 100ul each single use 24x100ul. It has protease or protease and phosphatase inhibitors. With or without EDTA and The identity and concentration of each component is fully disclosed.
http://www.piercenet.com/browse.cfm?fldID=02040806
We use compleate EDTA-free from Roche plus NaF, PMSF, NaVO3 and Mg132. Works fine with very labile proteins such ACC syntases.
We use an EDTA-free protease inhibitor cocktail from Sigma (P8340) with PMSF and sodium orthovanadate added, in order to make lysates for immunoblotting procedures. It works well for all of our proteins of interest, including for studies of phosphorylation status, due to the added sodium orthovanadate.
Combining PhosStop phosphatase inhibitor and cOmplete protease inhibitor both from Roche is the key to keep the phosphorylation status of your protein withtout having degradation. We produce 10X stock solution for both these products and keep them at -20Celcius. They are ready to use when you buy and you just need to dilute a tablet to get your stock solution.
When harvesting proteins, we combine both, then dilute with PBS 1X and we get our proteins.
Hi, Sergio,
Roche inhibitors are known to be the best among others. All my colleagues recommend these tablets too.
Our lab also uses Roche Complete protease and PhosStop phosphatase inhibitors. Very easy to use.
Cocktail-added RIPA buffer is put into -20C freezer for next use. So do you add FRESH cocktail inhibitor into this cocktail-added RIPA buffer? Thanks.
RIPA with complete and phosphoSTOP can be frozen and kept at least for a couple of month, I have even used 2 years old RIPA for detection of active protein kinase by western blotting- But it might not work for all type of assay.
As Jean-Marc indicated, I recommend Roche product call Mini!! Dissolve the tab in extraction buffer and use it.
Good luck.
Like many others, I have mostly used Roche CompleteMini protease inhibitor tablets along with Roche PhoStop, but lately also have used (successfully) ProteaseArrest from G Biosciences (they have with and w/o EDTA), adding Na orthovanadate and NaF as phosphatase inhibitors.
I am using Protease inhibitor cocktail and phosphatase inhibitor cocktail from SIGMA and works fine. I am not sure whether you can stored at -20 the RIPA buffer aliqouts containing these inhibitors cocktails. I always used them fresh
I have used products from Roche, Sigma and currently HALT from thermo fisher. In general any combination of a complete protease inhibitor tablet and phosphatase inhibitor cocktail should work with you big name phosphoproteins (erk, Akt, p38 ikb). Either of these work fine. We make up 10 ml lysis buffer containing protease and phosphatase inhibitors at at time. we can generally keep it from 2 weeks to a month at 4oC with no notable fall-off. Main difference is roche is in a tablet and thermo comes in a liquid form. FYI, I have done similar assays with no phosphatase inhibitors at all, which is acceptable if you run your gels soon after isolating lysates. Hope this helps someone
For those of you that use the Roche mini products, one tablet of protease and phosphatase inhibitor/10ml of lysis buffer. Do you make up a stock of this inhibitor cocktail and how?
This thread is old, but it comes up in a google search, so here is my 2c.
I use Roche's COMPLETE in HeLa and E coli extracts; they are soluble in aqueous buffers. For small reactions like IPs, 0.1-1 ml in volume, an entire tablet is too much. So I crush a tablet and aliquot in 1 mg bits of powder in 0.5 ml eppendroves and store at -80. The tablets are very easy to crush by rolling with a glass tube.
The Pierce (now FIsher, ugh) analogue does not appear to be soluble in aqueous buffers despite their claims.
If anyone has a recommendation for an analogue of Roche at a better price that is water-soluble, would be great to know!
Our lab have used " Xpert Protease Inhibitor Cocktail Solution(100X) " from gendepot and satisfied w/ it.
I wonder the condition of " Xpert Phosphatase Inhibitor Cocktail Solution(100X)" any one have used?
Very helpful thread! I am going for the cOmplete™, EDTA-free Protease Inhibitor Cocktail and Roche PhosSTOP™. Thanks Sergio for asking, thanks everyone for contributing.
some protease inh cocktail are in DMSO ready to use and I believe nothing can happen ..old drugs are more stable than commonly believed..dealing about pharmaceuticals ..see Article A long‐time stability study of 50 drug substances representi...
so stability is more a marketting point than a real...danger...but yes, nowadays, every lab reagent come as ready,kit to use and people forget their training 😱 so apart from PMSF which is demonstrated to be unstable in aqueus buffer..(pmsf is not included in tablet and I am adding it from 100mM stock solution in ethanol)
Adam Lafferty If you do find the answer to this question, please let me know! I am struggling with this too! Thanks!
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