Using Antibodies in Nematodes

As mentioned in my last blog post, we are performing a comparative study of maturing oocytes in the nematode species R. sp. SB 347 and C. elegans. To do so, we use immunofluorescence and microscopy techniques to observe protein presence and location in the developing oocytes. Our primary method of immunofluorescent staining is using antibodies to tag certain molecules. In the immune system, antibodies are used to tag foreign molecules for destruction. For instance, when a virus attacks the immune system, certain antibodies will recognize the foreign threat and mark it for destruction by the rest of the immune system. To do this, antibodies recognize and bind to a specific binding site on a foreign molecule.

Scientists have taken advantage of this natural process of specific binding and tagging to develop markers for research purposes (Alberts et al., 2015). In Shakes’ Lab, we stain nematode gonads using antibodies against our protein of interest. We use this antibody binding capability to mark different proteins and molecules along the gonads. These antibodies are coupled with a fluorescent molecule to make them viewable under a microscope. By adding antibodies to our samples and then viewing these samples under an epifluorescent microscope, we can observe the presence and location of different proteins along the entire germline. FBF is an example of an antibody that we use to study protein activity. I’ve been using an antibody against FBF to determine FBF protein presence and location in R. sp. SB347 gonads. The FBF antibody recognizes and binds to the FBF protein when it is suppressing specialization of germline stem cells (Crittenden et al., 2002).

Sometimes, an antibody cannot cross-react with the same protein between two species. This was the case with FBF in C. elegans and R. sp. SB347. As a result, Shakes’ Lab developed a new FBF antibody that is compatible with R. sp SB347. These previous FBF studies revealed a variety of patterns in the gonads of R. sp. SB347 (Lin et al., unpublished studies). This summer, we performed the study again under different experimental conditions in attempts to enhance the precision of the antibody binding. As with previous studies, our replicated experiment yielded variable FBF patterns.

One such pattern observed was complex FBF presence within the germline. In this model, FBF positive staining was found in the mitotic region, as expected. However, there was also a surprising finding of staining in the meiotic region. More details on this finding can be found in my first blog post. Another interesting model from our experiments was variability in FBF staining in the most proximal oocyte. I will explain this pattern in my final blog post. These patterns suggest that the FBF protein is playing a role in not only maintaining mitosis of stem cells, but also in initiating oocyte maturation.

 

References:

Alberts, Bruce, et al. (2015). Molecular Biology of the Cell (6th ed.). New York: Garland Science.

Crittenden, Sarah L., et al. (2002). A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans. Nature 417.6889 (2002): 660-663.

 

Speak Your Mind