Purification of microtubule associated protein MAP1B from bovine brain: MAP1B binds to microtubules but not to microfilaments.
A simple procedure for the purification of MAP1B from bovine brain is described. The procedure requires two ion-exchange chromatographic steps and results in > 95% pure MAP1B with a typical recovery of about 25-30 mg/kg of brain tissue. SDS-PAGE analysis of the purified protein shows that it is composed of a high molecular mass (330kDa) heavy chain and two low molecular mass (32kDa and 18kDa) associated light chains. The estimated stoichiometry of heavy chain:light chain is 1:2 and 1:0.2 mole/mole protein for the 32kDa and 18kDa light chains respectively. Western blotting, using monospecific monoclonal antibodies, shows that only the heavy chain is recognised by the anti-MAP1B antibody and is not immunostained by either the MAP1A or MAP2 monoclonal antibodies. Purified MAP1B binds efficiently to both unpolymerised tubulin and polymerised tubulin and co-sediments with taxol-stabilised microtubules. Co-incubation experiments show that MAP2 can compete with MAP1B binding to microtubules, indicating common or overlapping sites. However, MAP1B binds to neither G-actin nor F-actin nor co-sediments with F-actin, suggesting that it is not an actin-binding protein.
Pedrotti
B
B
Department of Biology, University of Milan, Italy.
Islam
K
K
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003599:Cytoskeleton / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; C085284:microtubule-associated protein 1B