Mammalian Phosphatase inhibitors - (Jul/17/2008 )

Hi all, I've been attempting to detect the phosphorylation state of various proteins for quite some time now, and I've been using this cocktail that I made myself from reading about the contents of various commercially available cocktails.


Sodium Fluoride 10 mM (Acid Phosphatases)
Sodium Orthovanadate 1 mM (Tyr Phosphatases, Alkaline Phosphatases)
Sodium Pyrophosphate, decahydrate 2 mM (Set/Thr phosphatases)
beta-glycerophosphate 2 mM (Ser/Thr phosphatases)

these are the final concentrations in my lysis buffer.

The problem is I always seem to have trouble detecting my phospho-antigens. Sometimes they are easy to pick up, but the vast majority of the time I have trouble detecting them. I don't believe it is an issue with the cellular responses to my treatments, since these biochemical events are documented in the literature.

Can I just double or triple the amount of this phosphatase inhibitor cocktail I'm using? I don't see why this would have a detrimental effect on protein recovery during lysis, but maybe there is something I'm overlooking.

Thanks,

Hector

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1. I would suggest using commercial preps.

2. Failing that, yes, you can use at 2X without any problem. I have used commecial preps at 2X of what was suggested, can not be sure of your particular recipes.

3. Sample prep issues should be corrected. Timing.

4. Check these links for tips and try some more keywords:
http://search.vadlo.com/b/q?sn=158621799&a...bitor&rel=0
http://search.vadlo.com/b/q?sn=158621799&a...stern&rel=0

..

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Thanks for the advice. I have ordered a commercial cocktail with different inhibitors than I'm currently using.

But I just realized I may have been doing something foolish - in my attempt to concentrate my cell lysates I may have been using an inadequate amount of inhibitors relative to the amount of phosphatases present, because I was trying to collect a large number of cells in a relatively small volume of lysis buffer (and of course, a limited absolute amount of inhibitors!).

could that have been a problem?

H

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There is the problem right there. 'm glad that you took effort to write down your own troubleshooting here.

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Until last year I also did my own inhibitor cocktails, but then Roche brought out their inhibitor tablets... I tried and am really happy with them - no more mixing and working with toxic DMSO stock solutions, just add the tablet or a part of the aliquoted stock solution... and most important: they really WORK more effective than my own cocktails - I made some comparisons by western blotting.
I can only recommend PhosStop from Roche. These tablets are easy to use and work perfectively and reproducible which you can not guarantee with self made cocktails.
Maybe they still have their samples available ... just have a look on their webpage.

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PhosStop.....so it comes in tablet form.

It says one tablet is good enough for 10 mL of lysis buffer on their website......I rarely make up that much for an experiment, since I like to work with fresh buffer. How do you go about working with these tablets? is it easy to break off 1/4 tablet? or do you just make up 10 mL and store it in the fridge? I don't imagine that'd be a very good idea if to keep this at 4 degrees for very long, especially if you've added protease inhibitors.

H

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Well, they guarantee that the stock the solution is stable up to 6 months at -20°C... so I did (stored it for up to 4 months) and it still worked fine (used it in Western Blot)... I think they have very stable inhibitors

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