Cell separation : a practical approach by Derek Fisher, Gillian E. Francis, David Rickwood

By Derek Fisher, Gillian E. Francis, David Rickwood

Setting apart particular cells or fractionating attainable mobile populations is a vital step of the various ideas used throughout quite a lot of organic and similar disciplines. this article goals to notify these operating in all components of the organic sciences of the tools that are to be had to them and that's more likely to be the main necessary. The contributing authors have supplied a complete consultant to the tools used to organize mobilephone suspensions and to split attainable cells reckoning on their varied features - equivalent to measurement, density, floor cost and immunological identification. every one bankruptcy bargains direct useful assistance for a number of the innovations and describes the benefits and barriers of every. Reproducible protocols, tricks and information for fulfillment and troubleshooting feedback are supplied

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Alternatively, the target protein could be transferred and immobilized on a matrix such as nitrocellulose where additional analytical procedures may be performed. This chapter describes methods to tag the target protein, that is usually present as a minor component of a cell lysate, and then specifi- Labeling Cells and Proteins cally follow it. General methods are presented for metabolically labeling live cells, and for labeling proteins located on the cell surface. In addition, the Chloramine T method of labeling a pure protein in solution is presented.

Labeling Cells and Proteins This method is a double immunoaffinity precipitation. First the target protein is immunoprecipitated with specific antibody; then if it is biotinylated, it is precipitated with streptavidin-agarose. 1mM CaCl2, 1mM MgCl2 in PBS Transwell inserts for 6 well plates: commercially available from many sources (Costar, Falcon, Nunc). 6 well plates Expre35S35S labeling mix (Perkin Elmer) 10% SDS (Appendix C) 1% SDS Glycine: 50mM in PBS+ Lysis buffer: 1% Triton X-100 in PBS Protein A-Sepharose suspension: Appendix C Streptavidin-agarose beads: (Pierce) Microcentrifuge and tubes Scalpel 2¥ SDS sample buffer: (Appendix C) 1.

Alternatively, the signal can be detected using phosphorimaging (Chapter 7). Comments: Labeling surface proteins with iodine forms a covalent bond between tyrosine residues and iodine. Labeling cells with NHSLC-biotin creates a covalent bond with lysine and arginine residues and biotin. This technique can be modified to detect the cell surface target protein by labeling with NHS-LC-biotin, performing immunoprecipitation, running SDS-PAGE, transferring to a membrane and visualizing the biotinylated protein on the blot with avidin coupled to a detection system.

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