I packaged some lentivirus to infect the cell line for stable cell lines. The antibiotic I used is puromycin. My cell lines can survive in a medium with 2ug/ml puromycin. However, when I detect my target protein by WB, I can't find any band in the NC membrane.
How should I proceed? Should I select it again? Can you share any suggestions?
1.PCR to check the PuroR fragment before you check protein
2.titre your cell line with Puromycin before you do selection
3. when you do puromycin selection, do a parallel experiment of cell without virus infection, make sure cells without PuroR are all dead after selection
1. Are you using a positive control for your WB? (It's important to know that your antibody works.)
2. How long are you selecting? Are all the cells on your control (untransfected) plate dead?
3. Try RT-PCR to check if your cells are making the mRNA for your protein of interest.
4. Sequence the vector you're using to make sure there are no mutations in the protein coding region.
Thank you very much, ever body. I have done a kill curve befor my selecting.The parallel experiments were made too. I didn't do a positive control for my WB this time. However I have detected my bands using the same antibody three weeks ago.I think the plasmids and the antibody works. I don't think the RT-PCR is meaningful if I can't detect the protein. Anybody has other suggestions?
Low titer of your lentivirus may cause low copy number insertion end up reduced the expression of your protein
also maybe the gene expression got silencing by some epigentic mechanism.
RT-PCR is important precisely *because* you can't detect protein: it will help you determine whether protein is being translated but degraded, or just not being made in the first place. If you don't want to do RT-PCR, you could also incubate your infected cells with cyclohexamide and then try your WB again.
Hello, did you figure out your question? Because I met the same problem right now. If so, please share your experience. Thank you.
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