Purification of monomeric actin

Richard P. Grant rgrant at netscape.net
Tue Feb 8 09:15:21 EST 2000


In article <Yw3o5VAeL$n4EABv at genesys.demon.co.uk>, "Dr. Duncan Clark" 
<Duncan at genesys.demon.co.uk> wrote:

> Does anyone have a protocol (or reference) for purification of gram
> quantities of monomeric or G actin.
> 

Hi Duncan,

Try the method at 
http://www.users.waitrose.com/~rkrs/thesis/2matmeth.htm#216

which is scalable.

The appropriate refs are Feuer et al, 1948, Spudich & Watt, 1971 and 
MacLean-Fletcher & Pollard, 1980.  I can dig out the full citations if 
you can't find these on Medline - 
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed

If you want 'dirty' G-actin (i.e. monomeric) then omit the S-100 step, 
although this *will* leave a known contaminant in there (check the 
MacLean ref).

I used to get 80 g acetone powder from 2 rabbits, and 20 mg actin/g 
powder, so that's a gram and a half.  :-))

Note that polymerization is a key step in the purification, but it is 
easy (if tedious) to obtain G-actin - just dialyse the filamentous (F) 
actin against 10 mM Tris-Cl pH 8, 0.2 mM ATP, 0.2 mM CaCl2, 0.2 mM DTT 
for two days . . .

HTH,

Richard

-- 
Richard P. Grant MA DPhil          
Structural Studies Group, MRC-LMB
http://www2.mrc-lmb.cam.ac.uk/personal/rpg/index.html
Please reply to rpg 'at' mrc-lmb.cam.ac.uk




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