Hi, Gosia, 



I modified the protocol and increased both purity and reproduction. The protocol was written in detail in my paper  BBRC. 2000 Apr 29;271(1):75-81.



(Liu X1, Ito K, Lee RJ, Uyeda TQ).  You may take a look at it.  Good luck. 



Xiong Liu 





-----Original Message-----

From: Gosia Poczopko [mailto:[log in to unmask]] 

Sent: Monday, February 02, 2015 11:03 AM

To: [log in to unmask]

Subject: [DICTY] problem with myosin-II 1-day purification



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]

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