The cell line used is mouse fibroblast cell line. Retrovirus carring a human gene with an RFP tag is used for transduction. Then puro selection and RFP+ sorting.
Cells transduced show RFP+ and puro resistant. The RNA level of the overexpressed cells is largely increased compared with the control. However, I cannot detect any protein level using either anti-RFP or anti-myprotein in western blot.
I ask a lot of people. Some say that the protein is rapidly degraded. If it is so, how can i know if it is rapidly degraded, as RFP is still there?
Any suggestions for detection?
Did you consider the mean fluorescence intensity of the RFP+ cells? May be you can design a time point study of the RFP+ cells post sorting then you may have a definitive conclusion of the RFP degradation. However, if you know the half life of the RFP protein that may explain the low level of RF protein expression observed in your cells. I hope this help.
Can you see the RFP+ cells under microscope? If no, the protein is rapidly degraded for sure. If yes, but still no bind, there maybe several reasons:
1. Have you checked the antibodies? You need at least a positive control for your antibodies.
2. Did you use the right way to collect the protein lysates? How about your loading control, such as GAPDH, or Actin?
3. Have you considered the epigenetic changes, such as protein methylation or unmethylation? Proteins can usually be silenced when epigenetic changes happen.
Hope this information may help you find the problems.
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