View source for In-vitro kinetochore-microtubule binding assays (Stukenberg lab)

Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]


link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]



In vitro Kinetochore-Microtubule Binding Assays


Monolayers of Xenopus tissue culture cells (XTC) were grown in 66% Leibovitz L-15 media containing 10% fetal calf serum, 1mM sodium pyruvate and antibiotics at ~21°C.  

Mitotic chromosomes were purified from XTC cells after a 16 hour block in 10µg/ml vinblastine (Sigma-Aldrich).  Chromosomes were purified as previously described with minor modifications (Wordeman et al., 1991).  

Experiments were preformed essentially as described (Mitchison and Kirschner, 1985).  

Purified mitotic chromosomes were diluted in reactions containing 10 µM tubulin in BRB80 buffer plus 1mM GTP.  Reactions were incubated at 37°C for 15 minutes in a water bath and were fixed with 10mM EGS (ethyleneglycol-bis-succinimidyl-succinate) dissolved in BRB80 for 10 minutes at 37°C.  

Reactions were layered onto a 30% glycerol cushion prepared in BRB80 and chromosome-microtubule complexes were pelleted through the cushion onto poly-l-lysine coated coverlslips.  

Coverslips were post fixed with ice-cold methanol for 5 minutes at 4°C, and processed for immunofluorescence with the DM1? anti-tubulin antibody (covalently conjugated to FITC).  

Antibody inhibition reactions were preformed by adding affinity purified antibodies to reactions to block the function of the kinetochore proteins xCep57 and xNdc80.  

P50/Dynamitin was purified as described (Wittmann and Hyman, 1999) and added to the reaction to inhibit the activity of Dynein/Dynactin at the kinetochore.  
 
 
 
  References
 
      1.    Mitchison, T.J. and M.W.Kirschner. 1985. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J. Cell Biol. 101:766-777.
      2.    Wittmann, T. and T.Hyman. 1999. Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes. Methods Cell Biol. 61:137-143.
      3.    Wordeman, L., E.R.Steuer, M.P.Sheetz, and T.Mitchison. 1991. Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes. J. Cell Biol. 114:285-294.