Abstract
The rlp-1 gene is a member of the rab small GTP-binding protein family. Using the yeast two hybrid analysis, our laboratory has previously identified a cDNA clone encoding a small heat shock protein like protein that we named SHSPL. To further explore the subcellular location and hence the physiological function of these proteins, we have initiated experiments to generate monoclonal antibody specific to these proteins. A hybridoma H569F was obtained that can produce antibody specifically recognize the SHSPL. Immunofluorescence analysis of the SHSPL has demonstrated that the protein is distributed throughout the cytoplasm of the HeLa cells. This finding is similar to that of the HSP27, another member of the small heat-shock protein family. The SHSPL was also found to present in rat heart, kidney, brain and skeletal muscle. In a parallel experiment, we also obtained a hybridoma named R423G that produced antibody specifically detects RLP-1. Using a number of truncated forms of RLP-1, the epitope recognized by the antibody can be mapped to the C-terminal half of the RLP-1 protein. The RLP-1 in transiently transfected HeLa cells can be localized in cytoplasm membrane as well as in small granules. Both of the antibodies were found to be highly sensitive. Less than 1 ng of the antigens can be detected with the corresponding antibobies using either ELISA or Western analysis. In addition, both antibodies can be precipitated by Protein A-agarose suggesting that they belong to a subclass of IgG. We believe that the monoclonal antibodies should prove valuable for their application to analysis the characteristics and function of the SHSPL and RLP-1.