limited proteolysis

Jonathan B. Marder marder at agri.huji.ac.il
Wed May 28 11:51:26 EST 1997


In article <338B4EA0.1E32 at aecom.yu.edu>, Alan Schoenfeld <aschoen at aecom.yu.edu> wrote:
>I want to take a metabolically labeled band from a gel by either slicing
>the band out of the gel or by transfering it to a membrane and then do
>limited proteolysis (V8, chymotrypsin, etc.) on the gel slice or
>membrane.  Can anyone give me a protocol to do this?  
Take a look at Cleveland DW et al. (1977) JBC 252, 1102-1108.

>If I cut from a
>gel, can I dry the gel first (I need to find my band by
>autoradiograph)?  Which enzymes are best/cheapest?  Thanks.
I have experience doing exactly this. You will find the details in 
Marder JB et al. (1986) Methods in Enzymology Vol 118, 384-396.

BTW cheap dirty enzymes work great. If you use them at limiting concentration, 
they will often pick up just 1 or 2 particularly suscptible sites in the 
substrate protein. I had success with Sigma Bromelain - 40% protein, 60% crud.



Jonathan B. Marder             ,      Department of Agricultural Botany
E-mail: MARDER at agri.huji.ac.il |      The Hebrew University of Jerusalem
Phone: (08 or +9728) 9481918   | /\/  Faculty of Agriculture
Fax:   (08 or +9728) 9467763   |/  \  P.O.Box 12, Rehovot 76100, ISRAEL
     http://www.agri.huji.ac.il/~marder



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