Ecoli protein extracts

rpgrant at molbiol.ox.ac.uk rpgrant at molbiol.ox.ac.uk
Fri Apr 2 02:35:29 EST 1993


In article <9304012319.AA15798 at audrey.lmf.org>, jim at AUDREY.LMF.ORG (Jim Gale) writes:
> 
> I need to make crude protein extracts from Ecoli to assay for 
> protein activity of a gene cloned into Bluescript SK- (isolated from
> a cDNA expression library). What are your favorite methods for
> making 'functional' protein extracts from Ecoli (ie no denaturents
> used in prep that might kill protein activity). Also, this
> plasmid is inducible with ITPG.
> 

Jim,

I use this method when I'm making fusion proteins from Ecoli.

Dilute 10ml of an overnight culture into 200ml media.
Grow at 37C, 1h.  Induce with IPTG, grow at 37C, ~3h.

Pellet cells at 5,500 x g <6k rpm in JA14> at 2C, 15'.
Resuspend pellet thoroughly in 25ml COLD
20mM Tris pH 7.4
200mM NaCl
1mM EDTA
(+ 1mM DTT, 1mM azide, 0.5mM PMSF added just before use)

Snap freeze on dry ice/EtOH for 15 - 30'.  Thaw in 37C waterbath for a few 
minutes (should get a nice "crack").  Repeat if you want.

Sonicate ON ICE for 4 bursts of ~15s, leaving 30s between each blast.
         ^^^^^^  this is important
Use a probe sonicator on max.
Spin at 12k x g <10k rpm in JA 20> at 2C, 15'.

The supernatant is your crude protein extract.

This protocol is adapted from NEB's fusion protein prep.
Standard disclaimers.....
Richard
-- 
R. P. Grant    <><                     rpgrant at molbiol.ox.ac.uk
Sir William Dunn School of Pathology   Fax. +44 865 275556
University of Oxford, UK.              Tel. +44 865 275565

Never knowingly undersold!



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