protein extraction

[Dr Engelbert Buxbaum]

Am 19.07.2009, 16:47 Uhr, schrieb riccardo senter <ricc_wparma from yahoo.it>:

> hi!
> i'm a new entry.
> i need to extract proteins from PBMC in order to perform a western blot  
> and a functional assay of iNOS, which is a cytosolic homodimeric protein.
> which is the best extraction protocol?
>
> Another question: can i use PBMC pellets that have been storing for  
> several years at -20°?

I would simply start with a hypotonic buffer (20 mM HEPES-KOH pH 7 or the  
like) with the usual protease inhibitor coctail and pass the cells through  
a fine (27G) needle a couple of times. Then spin down nuclei, membranes  
and the like in an Eppendorf centrifuge (refrigerated). 2 min on full  
speed should do it. The supernatant contains the soluble proteins.

If that does not work, you need to add detergents to your buffer. A very  
effective way is to use CTAB, then use cationic electrophoresis and  
eastern blotting for detection (Anal. Biochem. 314 (2003) 70-6). CTAB is  
even more effective in solubilisation than SDS, yet at the same time so  
gentle that it is often possible to run zymograms.

As to the old samples, if they have been prepared with protease  
inhibitors, snap-frozen, constantly stored at low temperatures (no  
self-defrosting fridges!) and if you want to detect only protein, not  
post-translational modification or activity, it should probably work,  
especially if you have samples of known target concentration stored in the  
same way as controls.