CNBr Cleavage of soluble proteins

Tan T.M.Franklin at bham.ac.uk
Fri Jun 14 05:48:28 EST 1996


In article <4p53pt$a60 at cnn.Princeton.EDU>, micky at berthaw.princeton.edu (Michael West) says:
>
>I am gearing up to do CNBr cleavage of candidates from a library of 
>fusion proteins.  I am cleaving a leader sequence from the C-terminal
>end and a antibody and His-tag from the N-terminal end.  The resulting
>protein is ~8kd and should be soluble.
>
>I would like to hear from anyone who has successfully performed
>this type of cleavage and/or I would like to find a good references 
>on the subject.
>
>Thanks,
>Michael
>-- 
>"...none of the tears that we cry in sorrow or rage
>        Can make any difference, or turn back the page..." -DG
> ************************************************************************                                                 
>Michael West                            micky at berthaw.princeton.edu 


Hi Michael
	I don't know if this is any help but I did a CNBr digest at the start of
the year. I had an N terminal blocked protein which I wanted to protein 
sequence. What I did was:-
Run 1mg of my protein on  Mini SDS PAGE
Stain with comassie blue ect. and cut out the relervant band.
Put gel slice in a microfugetube, but stick it to the wall near the top.
Place 200ul 70% formic acid in the bottom (don't let the gel touch) purge with
nitrogen and add 4 crystals of CNBr Purge with nitrogen.
Seal the tube and keep in the dark for 24hrs at room temp.
Remove gel slice and rinse in ddH2O and wedge onto the top of a new gel.
Half fill well with running buffer and alow protein 30 mins to diffuse.
Run gel at a low voltage (30v mini) to let protein enter the gel.
Run gel and treat as normal.

I hope this is some help
All the best Tan.



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