Katie Munechika / Dr. Ethel Cesarman - Week 8

 This last week, I have spent most of the time finishing up my research project in the lab. Besides the IP’s using the LY19 cells that I reported on in the last post, I also performed the IP’s with the V5-tagged H1E cells that we had, just to compare even though I did not use a V5 antibody for the IP. I ended up with similar results showing that H1C and H1E were unable to be pulled down. For these IP’s however, I was even unable to see a band for H1E in the input or unbound lanes, which I was able to see in the LY19 cell IP’s. I am wondering if the alteration of the V5 tag in the H1E protein may be hindering its expression in some way, and may also be contributing to why the V5 antibody is not working properly. I also checked the two control IP’s that I did using non-specific mouse IgG and rabbit IgG antibodies. For these I was expecting to see only bands in the input and unbound and nothing in the elution, so these worked as expected. Since I identified the antibody conjugation to the beads as the main issue, the next steps would be to optimize this process. I am not here long enough to work on this, but factors that could be changed would be trying different antibody to bead ratios or adjusting the amount of time they are incubated together. There is also a possibility that the beads I used were a bad batch or the antibodies were not compatible with that particular type of bead for some reason. I believe it would be worth the time to try a different CRISPR experiment design and attempt generating tagged H1 isoform cell lines. This would remove a lot of unknown variables and simplify the experiment as long as we are able to confirm that the tag efficiently binds to the antibody.

The post doc that I have been working on this project with is also leaving the lab soon, so I have been helping with wrapping up this project. This has involved going through the frozen samples and cell stocks and updating the inventory list. To finish up the mouse work, I helped again with collecting and processing spleens as well as bone marrow from the mice. For the spleens, we crush them, lyse the red blood cells, and extract the B-cells for flow cytometry analysis. For the bone marrow, we can cut a cross section in the bone and centrifuge it to pull out the bone marrow. Then the pellet of bone marrow is resuspended in a PBS-based buffer and saved for future bone marrow transplant experiments.

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