Phospholipase A2: microinjection and cell localization techniques

Bar-Sagi, D. (1991) Phospholipase A2: microinjection and cell localization techniques. Methods in Enzymology, 197. pp. 269-279. ISSN 012182098X

URL: http://www.ncbi.nlm.nih.gov/pubmed/2051920
DOI: 10.1016/0076-6879(91)97152-O

Abstract

This chapter focuses on two approaches, microinjection and cell localization, to analyze the biological properties of cellular phospholipase A2 (PLA2). As both approaches rely primarily on the availability of anti-PLA2 antibodies, methods for obtaining suitable anti-PLA2 antibodies are also included. The microinjection technique is a valuable tool for the assay of the functional significance of cellular proteins. This technique allows one to modulate the level and/or activity of a particular protein by introducing defined amounts of the protein or antibodies directed against the protein into the cells. In view of the paucity of specific PLA2 inhibitors, microinjection of inhibitory anti-PLA 2 antibodies makes this approach the method of choice to assay the role of phospholipase A2 in various cellular processes. The procedure utilizes a glass capillary needle filled with the substance to be injected into the cell, a micromanipulator to place the needle into the cell, and a phase-contrast microscope to allow visualization of the injection process. The analytical potential of microinjection experiments depends on two parameters: (1) the ability to positively identify injected cells, and (2) the availability of a single cell assay that can be monitored visually. For convenience, cells are grown on coverslips, which can then be processed and mounted on slides for observation. In the case of cells that adhere poorly to glass, adhesion and cell growth can be improved by cleaning the coverslips with chromic acid or coating the surface with a positively charged polymer.

Item Type: Paper
Subjects: Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > phospholipase A
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date Deposited: 21 Dec 2015 16:59
Last Modified: 21 Dec 2015 16:59
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32120

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