Katie Munechika / Dr. Ethel Cesarman - Week 5

 For this week, I have determined next steps for performing immunoprecipitation based on the results of the western blot from last week. Observing the blot more closely, the H1E specific antibody worked well when the blot was overexposed. However, it was clear that the V5 tag was not working, as there was no band at all, even when overexposed. The H1E band in the V5-tagged H1E cells also had much lower signal than the non-altered LY19 lymphoma cells. It’s possible that the process of trying to add the V5 tag to the H1E gene may have inhibited its expression in some way. Although we confirmed that V5 was present in the H1E gene through sequencing, it could be that its position in the protein structure may not be a favorable spot for antibody binding or the structure itself may have been altered during translation which would prevent antibody binding, since the chromatogram reading from directly after the V5 sequence was messy and difficult to interpret.

According to these results, it seems that it will not be possible to successfully use V5 for the IP experiment, however, I may be able to use the H1 isoform specific antibodies directly. Even if the western blot signals for these antibodies were faint, mass spectrometry is extremely sensitive and should still be able to pick up the signal sufficiently. Moving away from the idea of using V5, I have decided to test the IP with the NSD2, H1C, and H1E antibodies. For this experiment, I still want to perform the IP using both cells lines (tagged and non-tagged) to see if it is possible to identify tagged H1 via mass spectrometry. For the negative control, I will bind non-specific IgG to the beads that we use for the pull-down, while the sample will use beads that are bound to the antibody of interest. Although this is not quite as strong of a control as using tagged versus non-tagged cells, it will be necessary since all LY19 cells contain our proteins of interest.

I also did some mouse work this week where I learned how to collect and process mouse spleens. The H1 mutations that we are most interested in have been shown to mainly affect germinal center (GC) B-cells. Since the spleen is the area where B-cells differentiation occurs, we collect spleens from immunized mice (which instigates B-cell development) and isolate the B-cells from the samples. Then further identification and characterization of the particular cells of interest can be done via flow cytometry.

My previous clinical experience has mostly been in the infectious diseases department, since the lab specializes in viral oncology. However, with my research project mainly focused on lymphoma, I wanted to learn more about this area on the clinical side. I reached out to Dr. Peter Martin in the hematology/oncology department who works specifically with lymphoma, and I am planning to meet with him next week to discuss shadowing opportunities.

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