Hi all,
I am trying to express several proteins linked to GFP in Jurkat T cells. I have prepared the DNA constructs (using pEGFP-C1) and have electroporated them into my cells. I selected with G418 for 2 weeks and sorted the surviving cells with flow cytometry to retain those that actually had GFP fluorescence (about 20% of the population). Jurkat T cells are difficult to transfect with lipid-based carriers.
To check and make sure that the cells are actually expressing my GFP-linked proteins of interest, I lysed some of each type of cells and ran the whole cell lysate on a gel, followed by Western blotting with anti-GFP. I was expecting each of my cell types to have a band of a different molecular weight (for example, the GFP-C terminal deletion protein should be about 45kDa and the GFP-full length should be about 80 kDa, and etc.) After developing the Western, I saw a band at about 70 kDa in each type of whole cell lysate that was detected by the GFP antibody. None of my constructs are supposed to be 70 kDa and they should all have visibly different molecular weights! I also ran a sample of untransfected cells and saw no band there, so it's definitely coming from the transfection...
I previously made an mCherry-tubulin construct and have successfully expressed it in Jurkat T cells using the same protocol (I can see the microtubules, so I know it works). Anyone have any insight as to what's going on with my GFP cells? I have no clue!
WonderRakeWoman
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