Dear Dictybase users,

I run into a problem during myosin-II purification from dicty and was 
hoping maybe someone here can help me.

I am using the 1-day protocol by Kathleen Ruppel (Ruppel et al. 1994, 
JCB. 269: 18773) that can be found on this website in the methods 
section. The method is based on the consecutive rounds of protein 
assembly and disassembly which depends on the salt concentration in the 
buffer. The method: 
http://dictybase.org/techniques/cytoskeleton/myosin_prep_1day.html

I got stuck at the step 23 (of the on-line version, it's step 12 in the 
word file) in which I am supposed to homogenise the pellet in 0.3-0.5 
vol/g cells of high-salt extraction buffer. This pellet contains the 
mysoin-II, which I am after. It seems I cannot resuspend the pellets. I 
tried using the glass douncer and made sure that the buffer is fresh. 
Judging from the SDS-PAGE I'm loosing majority of my protein in this 
step (myosin-II remains in the pellet and very little of it is present 
in supernatant that I'm processing further).

Do any of you have experience with this method? Is there a trick to 
homogenize the pellets in this step?

thanks for taking your time and have a great day,
Gosia

-- 
Gosia Poczopko

PhD student
Cellular and Molecular Biophysics (Schwille Lab)
Max Planck Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried/Munich
Germany

Phone: +49 (0) 89 8578 2969
e-mail: [log in to unmask]
http://www.biochem.mpg.de/en/rd/schwille/people