Lysing E.coli for protein analysis

Hernan Espinoza espinoza at cgl.ucsf.edu
Fri Nov 10 22:54:01 EST 1995


Duncan Clark <Duncan at genesys.demon.co.uk> writes:

>Hi folks,

>A simple problem. I am screening umpteen E.coli clones for expression of my 
>protein on a 3ml scale. I microfuge 1.5ml and resuspend in 100ul 10mM Tris pH 
>8.0.I then sonicate, microfuge, remove 10ul into 10ul 2 x cracking buffer, heat
>(stand in freshly microwaved water for 3 mins.) and load 10ul onto SDS PAGE gel
>Works fine however the sonication step is a pain because of the time it takes 
>and it heats the protein too much. If I put 100ul of cells into 100ul 2x 
>cracking buffer, heat then microfuge (10mins) the solution is still too 
>gelatinous to load ie the DNA/RNA will not spin down to leave me a nice 
>pipettable solution. I can get around this by sonicating before microfuging but 
>again this adds time. 

>Anyone have a simple alternative method.


	What is 2X "cracking buffer? Just curious. Here's what I do...

1) spin down  0.5ml cells

2) Resuspend in 200 microlitres 1X sample buffer = 62.5mM Tris pH6.8 
						   10% (v/v) glycerol
						   5% (v/v) 2-mercaptoethanol
						   2.3% SDS
						   0.01% (w/v) Bromophenol Blue

3) Vortex ~30sec (or less)

4) Place in Boiling water for 5 min

5) Spin 10' in a microfuge

6) Load 5 microliters per lane (for over-expressed proteins and westerns)

	Well...that's it.  It works pretty well for me, and I have a 
really high throughput.  IT will NOT tell you weather or not your 
protein is soluble/folded/etc., just that it is there.  Good luck!

-Hernan (espinoza at cgl.ucsf.edu)


>Duncan
>-- 
>-----------------------------------------------------------------------------
>My mind's made up. Don't confuse me with the facts!




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