By isabelle Vernos
Isabelle Vernos and a panel of hands-on specialists current their most efficient and reproducible ideas for the identity, purification, and characterization of the kinesin superfamily of microtubule-dependent cars. The tools variety from the main simple to the main subtle and contain step by step directions and wide cautionary notes to make sure experimental good fortune. one of the ways thought of are easy methods to convey and purify kinesins in numerous platforms, to represent microtubule-enhanced ATPase job and motility houses, and to check microtubule destabilizing task. entire and hugely functional, Kinesin Protocols makes on hand the entire key easy and state of the art equipment had to effectively examine the multifaceted international of kinesin-like proteins and to discover their many services.
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Extra info for Kinesin Protocols (Methods in Molecular Biology Vol 164)
298, 525–541. 6. , and Muhlrad, A. (1995) Immunochemical probing of the functional role of the 238–246 and 567–574 sequences of myosin heavy chain. Eur. J. Biochem. 232, 235–240. 7. , Perusinghe, N. , Moss, D. , Chen, X. , and Evans, W. H. (1996) Gap junction distribution and connexin expression in human breast. Exp. Cell. Res. 223, 29–38. 8. Cole, D. , Cande, W. , Baskin, R. , Skoufias, D. , Hogan, C. , and Scholey, J. M. (1992) Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.
Lysozyme. 14. Phosphocellulose: P11 (Whatman) (see Note 2). 15. DEAE resin: DEAE BioGel (Bio-Rad). 16. Sonifier: Branson model 450 with wide tip. 3. 1 mM (see Note 3). 1. Expression of Constructs 1. 8 L of fresh media in a 4-L flask. 5 h at 37°C. Expression of Kinesin in E. coli 45 2. 05 mM to induce expression of the recombinant protein and the cells are shaken for an additional 4–6 h at 37°C. 3. The cells are chilled on ice and collected by centrifugation for 6 min at 6000g (6000 rpm in Sorvall GS3 rotor) (see Note 4).
Subsequent sequence data revealed that the clones that hybridized to the degenerate oligo were almost exclusively kinesin-related proteins. Furthermore, the extent of crossreactivity of the purified plaques identified in the antiLASGE screen was tested with both anti-HIPYR and anti-LAGSE antibodies. Those plaques that did not crossreact with anti-HIPYR, even if they exhibited extremely strong anti-LAGSE crossreactivity, were not kinesin-related proteins. Hence, kinesin-related proteins exhibited some degree of crossreactivity with both anti-HIPYR and anti-LAGSE and reflected homology with DNA sequences derived from conserved regions of the kinesin motor domain.