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By isabelle Vernos
Isabelle Vernos and a panel of hands-on specialists current their most efficient and reproducible concepts for the identity, purification, and characterization of the kinesin superfamily of microtubule-dependent cars. The tools diversity from the main easy to the main subtle and comprise step by step directions and wide cautionary notes to make sure experimental good fortune. one of the techniques thought of are the right way to show and purify kinesins in several structures, to signify microtubule-enhanced ATPase job and motility homes, and to check microtubule destabilizing job. finished and hugely useful, Kinesin Protocols makes on hand the entire key uncomplicated and state-of-the-art tools had to effectively research the multifaceted international of kinesin-like proteins and to discover their many services.
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Collagen, laminin) that may simulate the in vivo matrix environment. Alternatively, the coverslips may be coated with a cell and tissue adhesive material. We have used and had success with Cell-Tak (Collaborative Biomedicals/Becton Dickinson Labware), Matrigel (Becton Dickinson Labware), and ProNectin F recombinant attachment factor (Stratagene). In Fig. 3, it appears as though the HIPYR antibody staining is more prominent than that of the LAGSE antibody. This is more a result of the imaging of these cells than actual levels of protein expression.
Biol. 13, 745–77. 21. Lane, D. and Harlow, E. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 22. , and Fritsch, E. F. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 23. Vallee, R. B. and Collins, C. A. (1986) Purification of microtubules and microtubule-associated proteins from sea urchin eggs and cultured mammalian cells using Taxol, and use of exogenous Taxol-stabilized brain microtubules for purifying microtubule-associated proteins.
Com. The principle of this protocol is to compare the expression of the set of kinesinlike motor proteins expressed in two distinct developmental (differentiation) stages or cell types, aiming to identify those that are uniquely expressed in each stage or that are subject to downregulation. , screening clones of one stage using the other stage cDNA pool as mixed probe and vice versa]). Those nonhybridizing clones will be good candidates for stage-specific expression (see example in Fig. 1B,C). Furthermore, RT-PCR for Kinesin-like Motors 19 this technique can also reveal clones that are developmentally downregulated resulting in weak hybridization, although such results must be interpreted with more caution because they may be artifacts resulting from inadequate probe labeling or they may simply reflect the relative abundance of a specific cDNA in the mixed pool of cDNAs used as a probe.
Kinesin Protocols (Methods in Molecular Biology Vol 164) by isabelle Vernos