Antibodies (Abs) are widely used to detect specific cellular components both in situ and in biochemical experiments aimed at defining protein-protein interactions, protein processing, and reversible protein modifications such as phosphorylation. Polyclonal Abs are relatively simple to make and are widely used in molecular and cellular studies. However, such reagents are limited in quantity and thus rarely made widely available. Monoclonal Abs are more complicated to make, but hybridomas secreting the monoclonal antibody can be passaged ad infinitum permitting the identical reagent to be used by many different research group over long periods of time. Polyclonal and monoclonal Abs are available commercially that are directed against a huge array of vertebrate cellular components. By contrast, few such commercial reagents are available for work with model organisms such as C. elegans probably because the market for these reagents is too small to make them commercially viable. I propose to develop a 'tool kit'consisting of monoclonal Abs directed against a series of proteins that label discrete cellular components and subcellular compartments of C. elegans. The compartments include organelles common to all cells (e.g. endoplasmic reticulum, the Golgi, endosomes), organelles of specialized cells (e.g.synaptic vesicles), subcellular structures common to all cells (e.g.nuclear membrane, centrosomes and caveoli) and components that label domains in the developing embryo, germline, and nervous system. The cellular components include macromolecular complexes (e.g.splicesomes, proteosomes, replication origin complexes) and common cellular proteins (e.gclathrin, kinesin, dynein and intermediate filaments). The Abs will be thoroughly characterized and made publically available through the Developmental Studies Hybridoma Bank. The tool kit will be extremely useful for C. elegans researchers. It will provide a set of standardized renewable reference antibody tools available to the entire community. Secondly, it will greatly facilitate localizing other cellular components by providing a large set of cellular markers that can be used coordinately with rabbit polyclonal antibodies for co-localization studies. Finally, it will stimulate the use of biochemical approaches, such as subcellular fractionation and immunoprecipitations, by providing means to detect specific cellular compartments in extracts and the presence of specific components in immunoprecipitates.
| Hadwiger, Gayla; Dour, Scott; Arur, Swathi et al. (2010) A monoclonal antibody toolkit for C. elegans. PLoS One 5:e10161 |
| Pant, Saumya; Sharma, Mahak; Patel, Kruti et al. (2009) AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nat Cell Biol 11:1399-410 |
