Gregory Lachaud
Generation of Affinity Purified Anti-sera to GLP-1 Protein
Mentor:  Dr. Tim Schedl, Department of Genetics, Washington University School of Medicine 

The primary purpose and ultimate goal of my lab research project was to generate affinity purified anti-sera to the GLP-1 protein found in Caenorhabditis elegans’ germline. C. elegans are small nematode round worms approximately one millimeter in length. The GLP-1 or germline proliferation-1 protein proves vital to the existence of c. elegans as a species. GLP-1 is essential for C. elegans germline stem cell proliferation and inhibits meiotic development. A primary reason for studying GLP-1 is because it is homologous to the Notch receptor, which is important to many different central systems of many different organisms.

GLP-1 fusion protein was cloned into a plasmid in E. coli therefore we first grew bacteria in Luria-Bertani broth. After the Optical Density of the solution reached a certain value we induced the bacteria with IPTG. The IPTG detaches the repressor embedded in the DNA plasmid of the bacteria and allows the GLP-1 fusion protein coding sequences to be transcribed into mRNA and then translated into the GLP-1 protein. Next we isolated only the pure GLP-1 protein we desired. To achieve this we coupled the protein to amylose resin and passed it through a filtering column. The column held the resin with the protein bound to it as we washed the column several times with different reagents. Once the column was washed of impurities we eluted the column through with glycine buffer, which due to its low pH detaches the protein and we collect the desired protein into fractions. Subsequently we check each faction for protein by running our obtained samples through a gel. Once the gel revealed which fractions had protein we pooled them together and bound them to guinea pig antibodies. Again we used the column to wash the protein bound to the antibody. Subsequently we elute the column with glycine and aliquot the antibodies into fractions. We then tested the fractions for evidence of strong antibodies and using our results we pooled the strongest fractions. The final process of washing and separating the antibodies is repeated several times because as the more the method is repeated the purer and more efficient your final antibodies will be. Now with our generated purified antibodies to the GLP-1 protein we expand our abilities for more experiments with GLP-1. These include Western blots and whole gonad staining in C. elegans.