Protease inhibition is essential for the quality of isoelectric focusing/2-D SDS-PAGE experiments. I recently used the 'Protease Inhibitor Cocktail for use with yeast and fungal extracts' from Sigma for IEF. When I diluted the cocktail 1/100 in my isoelectric focusing buffer (48 % (w/v) urea, 2 % (w/v) CHAPS, 0.5 % (v/v) ampholyte (pH 3-10) solution, 20 mM DTT, 0.002 % (w/v) bromophenolblue) it immediately formed a precipitate which could not be solubilized by vortexing. As a result, protease inhibition seemed not sufficient (medium to high molecular weight proteins were not present in the 2-D gel). What kind of protease inhibitor (or cocktail) do you use when you do an IEF/2-D experiment?
Thanks!
Hi
Generally i have not a problem with proteases, in IEF buffer: 6M Urea, 2 M thiourea, 4%CHAPS, 5mM TCEP (tris(2-carboxyethyl)phosphine, 0.5 % ampholytes, but for protein extraction in similar buffer i use cOmplete Protease Inhibitor Cocktail from ROCHE , there is no precipitation
http://lifescience.roche.com/shop/products/complete-mini-edta-free-3271382-1
I have never added this cocktail for IEF itself, but you can try.
Cheers.
Hi
Generally i have not a problem with proteases, in IEF buffer: 6M Urea, 2 M thiourea, 4%CHAPS, 5mM TCEP (tris(2-carboxyethyl)phosphine, 0.5 % ampholytes, but for protein extraction in similar buffer i use cOmplete Protease Inhibitor Cocktail from ROCHE , there is no precipitation
http://lifescience.roche.com/shop/products/complete-mini-edta-free-3271382-1
I have never added this cocktail for IEF itself, but you can try.
Cheers.
Many thanks for your suggestion, Wojciech!
Best regards, Christian
You have two options:
1. Work on ice under liquid nitrogen to deactivate proteases
2. Using of Protease inhibitors cocktail for four different types of protease.
I work without any proteases but one ice and under liquid nitrogen and found valuable results. You can find it in:
Electrophoresis. 2014 Dec;35(23):3331-8. doi: 10.1002/elps.201400273.
Hi!
I can suggest you a few things:
1. Use a cocktail protease inhibitors WITHOUT EDTA, that's Will allow to better focusing of proteins.
2. Review quantity of inhibitors are you using, Maybe proteins are precipitated because of a large proportion of cocktail.
3. Sonicate sample by 3 pulses of 20seconds on ice and them centrifugate it in cold conditions. Use soluble part of the sample.
4. And the most important...always preparate samples on ice.
Regards
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