I have been struggling lately to see expression of endogenous CCR7 in CEM cells as well as exogenous expression 72 hours post transfection of HEK293T cells. No signal (or weak signal) by flow cytometry and no signal by western blotting. I have several antibodies for detection of this GPCR but have very rarely gotten signal, which makes me wonder if it is all just an artifact of the antibody. I have decided to run a native gel since one of the antibodies Ive used has not been validated for WB and more than likely detects a folded region of the receptor. Any advice is greatly appreciated.
P.S. advice on overexpression of humanCCR7 in HEK293T cells is also welcome
GPCR are the membrane proteins which have a significant portion of their molecules in the very hydrophobic cell membrane environment. Therefore, these proteins are very hydrophobic and prone to aggregation without any detergents, and that's why they can not be used in a native gel. The most likely explanation for your observation is that your protein is aggregated into very high MW complexes, and did not enter even your stacking gel (the potential epitope might also be lost due to aggregation). Try to use SDS-gel under non-reduced conditions (without boiling) for WB. If you are lucky you might detect your protein (or use polyclonal Abs).
Hi Olga,
Since you want to detect a membrane (or membrane-bound) protein; I can Suggest some tricks for Western blotting...
(You are overexpressing the gene, so you must see it in any of the antibody you have. Please follow the bellow tricks..)
1. For Denaturation WB: Lyse the cells with 1% SDS lysis buffer (with other ingredients). Then run in 8-10% SDS-PAGE, transfer onto PVDF and then IMPORTANTLY- Do "Denaturation and renaturation steps" with 6M-Guanidine-HCL for O/N (You can find the protocol online). [To get a optimum condition for antibody-epitope interaction].
2. For Native WB: Lyse the cells with TRIS-buf with 10% glycerol (with other ingredients) by glass beads/sonication. Run it in 4% PAGE in TRIS-Boric acid-EDTA buffer for 2-3 hr at 4C. IMPORTANTLY- Soak the gel in 1%SDS-Running buffer for 30 min. Then transfer it on PVDF. Next follow the denaturation and renaturation steps as above. [But, membrane proteins tend to aggregate in such native condition].
However, if you are NOT interested in the native-complex and your protein is membrane bound; I will suggest for the "Denaturation WB". I follow the native protocol for a cytoplasmic huge protein complex (3MDa) detection.
OR..
Try Dot blot or immunofluroscence to detect since there is no loss of protein during the technique and proteins are in native confirmation. Or in worst case separate the cell membrane fraction and do a TCA precipitation and follow "1" (If you really want to detect you protein).
Good Luck.
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