4T1 cells (mouse) are transfected with human CCR2 plasmid, the cells are selected by G418 (0.8mg/ml), but western blot doesn't show the cells express CCR2.
Any ideas why this can happen?
Thanks!
It is difficult to answer this question as little information was given.
1) Are you sure that the expression plasmid you used is correctly constructed?
2) Usually I select these cells with 1mg/ml of G418. Did you check that control cells (not transfected with the plasmid bearing the resistance gene) were all killed by G418 at 0.8 mg/ml?
3) Do you have a positive control in your Western blot?
4) Is the protein you are trying to express toxic for the cells? In this case it can happen to obtain G418-resistant clones that do not express your protein of interest.
Yes, positive control for the Western is key and even the same cells that were transiently transfected with the plasmid would also show that the construct was expressing enough protein by Western since stable cell lines will likely express much less.
As for toxicity, I wouldn't conclude that there weren't toxicity issues unless you've seen others stably transfect that same ORF into those cells using a non-inducible system. I'm guessing that you weren't using an inducible system since you didn't mention it.
Lastly, did you linearize the vector such that the CCR2 cassette and neo cassette were together on the same fragment? Or did you use supercoiled plasmid?
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This will help
- check your antibody works, as suggested use a positive control.
- RT-PCR from transfected cells and sequence cDNA. This does not confirm that your protein is adequately expressed but does confirm that you have mRNA transcripts present
- Control transfection: something that always works in this system and is easy to detect
- FACS analysis on whole cells (if you have suitable antibody)
-make sure that during preparation of cells for WB that your protein is not degraded. use protease inhibitors if apppropriate.
- You could co-transfect a reporter plasmid with say SEAP or luciferase/GFP for confirmation of transfection efficiency
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