Katie Munechika / Dr. Ethel Cesarman - Week 4

 This week, I have been working on a few different areas in the lab. As mentioned last week, I was beginning to learn more about designing CRISPR Cas-9 experiments. Once I had a better understanding, I was able to design guide RNAs and donor oligos for V5 tagging of H1 isoforms in lymphoma cells. Since we already have cells with V5-tagged H1E at the N-terminal, I generated three other V5 knock-in designs: H1E V5 at the C-terminal, H1C V5 at the N-terminal and H1C V5 at the C-terminal. Although, I may not be here for the end product, the eventual goal is to compare the performance of these tags for pulling down the H1 isoforms during immunoprecipitation. To complete this task, I utilized the Alt-R HDR Design Tool on the IDT website to generate potential designs. Then, I compared the designs based on the on and off-target scores to determine which one would be best. Higher on-target scores indicate better efficiency for editing the target site, while high off-target scores indicate lower off-target effects on the cells. I also browsed through the list of off-target effects to ensure that none of them would heavily impact the functionality of the design.

Since we confirmed from the sequencing results that the V5 tag was present in the H1E N-terminal V5 cell line that we have, I re-did the failed western blot from last week. Since I was having trouble seeing the H1E/V5 band last time and the sequencing implied that the V5 tag was only present in some of the alleles, I decided to double the protein amount that I loaded into the gel. Additionally, I wanted to try blotting with a few different antibodies to better figure out what was present in our sample and try to troubleshoot the lack of H1E/V5 that happened last time. Besides testing both the old and new V5 antibodies we have, I also tested antibodies for total H1, isoform H1E, isoform H1C, a different histone H3, a histone interactor NSD2, and actin to confirm that protein extraction and digestion worked properly. I was able to see bands for NSD2, actin, total H1, H1C, and H1E, however I still do not see any bands for H1E/V5.

The next step after confirming the antibodies with the western blot is to actually perform the immunoprecipitation (IP) experiment. This is where we will pull down the H1 isoform of interest in complex with its interactors so that it can be analyzed via mass spectrometry. I have experience with IP’s from my lab in Ithaca, but I am usually used to dealing extranuclear proteins, whereas here, I am working with nuclear histone proteins. Therefore, I have been working on adapting my IP protocol to better serve this H1 project. For example, since we want to preserve protein-protein interactions during the IP, it is important to use a non-denaturing lysis buffer, however these types of buffers are not usually harsh enough to lyse the nuclear membrane along with the cell membrane. Considering our target is a nuclear protein, it is key that we are able to release it from the nucleus during cell lysis. As a result, it will be necessary to include some sort of mechanical disruption, such as sonication, along with the buffer to ensure that we can obtain our protein of interest. Furthermore, we will need to confirm that the mechanical disruption method is harsh enough to perform this task, but not so harsh that it will break the interactions.

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