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
