Blot overlay identification of microtubule-binding peptides from bovine brain.
Microtubule (MT)-binding peptides have been detected in homogenates of bovine brain tissue utilizing a blot overlay assay. Blots were prepared by the electrophoretic transfer to nitrocellulose of proteins separated on polyacrylamide gels. These blots were incubated with taxol stabilized MTs or tubulin, rinsed, and then fixed by air drying. About 17 soluble MT-associated proteins (MAPs) were identified by immunodetection of bound tubulin, including MAP2, kinesin, and tau. The interaction of MTs with these peptides appears to be specific, since MT binding can be displaced by a fluorescent tubulin analog, is competitively inhibited by the addition of exogenous brain MAPs, is decreased by raising the salt concentration, and is diminished by sodium dodecyl sulfate (SDS) denaturation. Only one protein (150 kDa) appears to have an interaction with MTs that is stable in high salt. The specificity of the binding on blots is further illustrated by the interaction of MTs with the MT-binding domains of MAP2 (32-35 kDa fragments) and kinesin (64 kDa fragment). Specific MT-binding peptides or domains can thus be isolated and characterized with this method, which requires little protein and is suitable for use with proteins that are either soluble or insoluble under physiological conditions.
Rozdzial
M M
MM
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347.
Neighbors
B W
BW
McIntosh
J R
JR
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D010449:Peptide Mapping; D014404:Tubulin / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D014404:Tubulin