Hi All,
I used Crispr/cas9 to knockout CISH in T cells. The T7EI assay showed the gene editing. However, when I used the Western blot to check the protein expression, I found the the bands with correct molecular weight were missing, but there were some unexpected new bands with higher molecular weight. Could anybody give me some suggestions what are these new bands? Thanks.
Hi Ying, are you working with pool or clones? Was western done with CISH-specific Ab? Indeed result is very strange as one might see degraded products. To suspect a fusion with other proteins? - That is not likely, especially because there multiple products. Confused... :)
Hi Dmitry,
Thanks for the response. I expanded the Tregs in vitro, did crispr, and harvested all the cells for WB. CISH is an intercellular staining. It's impossible for me to sort the cells. The antibody is a monoclonal anti-CISH that purchased from Cell Signaling (#8731). The results are weird. If there are some WT cells contamination, I should see some bands with correct size, but there are not. There are only two faint bands with higher molecular weight (CISH should have two bands). It seems that the protein is cut by crispr and relink to a bigger one??? I am very confused.
Hi Ying, from what you have told me, it seems you are working with pool of cells, not clones. So it should have some wt CISH protein present, and you are not seeing it, so I assume that your antibody may be not working? Try to change Ab, if available. Sequence your cells across the target area with NGS, to find out what is going on there. In general it is good to get individual clones, to work with, but it depends on what you are trying to do. :)
Hi Dmitry, thanks for your suggestions. I used scramble crRNA as a control, and run the wt cells and knockout cells on the gel side by side. The bands on the control lane are normal with correct molecular weight. So, I think the antibody is fine. NGS is a good idea. Maybe I can get an answer from sequence. I am working with Tregs, and really don't know how to get individual clones of Tregs. It will be highly appreciated if you can give me some suggestions. Thank you very much!
I see... Good that Ab is OK. Strange that you get that result on KO pool. Here is a paper that describes Tregs cloning: Article IL-17-producing human peripheral regulatory T cells retain s...
But I am not sure if you need to commit yourself to cloning if your pool gives this data. %)Keep me posted if you figure out what is going on!
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