I am recently using the MST (Microscale Thermophoresis) to study the interaction between the GPCR and small molecules. And I have established a stable cell line in HeLa overexpressed the GFP-tagged-GPCR, so I want to get the GFP-tagged-GPCR from cell lysate supernatant in order to perform the MST assay. But I cannot get enough GFP-tagged-GPCR in the cell lysate supernatant to conduct the MST assay.
Please give me some advice to improve the yeild in the supernatant. Thanks a lot.
Zhiyuan
Good point, Anne . But it is not convenient for me to conduct the sorting assay. Thank you very much all the same.
Recently, I am trying to get more GFP-tagged-GPCR in the supernatant, adding 0.1 to 0.5% TritonX-100 and NP-40. It seems that the fluorescent value can be enhanced about 20%. And i will try higher concentration of TritonX-100 and NP-40 , perhaps 1%.
But I donnot know whether such higher concentration of detergent will affect the interaction between the GPCR and small molecules.
Can you use the membrane fraction instead of whole cell lysate (is your GFP receptor localizing to the membrane like an endogenous GPCR?) This will exclude non-GPCR proteins and enrich for your GPCR, IF it is localizing correctly.
Kathleen,thank you for your advice. But MST assay should be conducted in the cell lysate supernatant instead of membrane fraction.
Hi Zhiyuan,
I will suggest firstly to check the expression level of the GFP-GPCR fusion protein in your stable cell line, double check by both of microscopy and western blot (WB), to ensure you picked a good clone.
If the expression level is fine, you cannot get enough amount of your target protein, the reason could be in the process of cell lysis. You can change the lysis buffer. It would be better to quantify the amount of your GFP fusion protein by WB in every step, in order to find out in which step you lose your protein and which lysis buffer is better. The concentration of NP40 at 0.5-1% in the lysisi buffer should be fine. Firstly, try to keep the target protein in the supernatant, not go to pellet. If you worry the interaction with small compound in the MST experiment, you can change buffer later with lower conc. of detergent.
If you only want to get enough GFP-fusion protein and study the interaction with compound by MST, another possibility is you can just do a transient overexpression, instead of the stable cell line. Because sometimes the transient overexpression can have an even higher yield of the target protein, compared with the stable cell line. Sure, you need to ensure the expression level here as well. For transient overexpression, it is better to use HEK293 cell line, because generally HEK293 is a better cell line to get a good overexpression of the target protein.
Hi Zhiyuan,
Most, probably all, GPCRs will denature in the detergents you are using and either precipitate, or will be non-functional in your assay. You could try using LMNG, a relatively new detergent that has been successfully used for crystallization and NMR. Otherwise the gold standard detergent is DDM.
Good luck,
Dan
Hi Zhuo，
Thanks very much for advice and analysis. I just want to get enough GFP-fusion-GPCR in order to conduct MST assay and it will be a good choice to perform transient transfecion . But now I am worried about the activity of GPCR just from cell lysate, just as Daniel have analysed.
Best
Zhiyuan
Hi Dan,
Thanks a lot for your suggestions.
Now I am wondering whether adding the detergent LMNG or DDM in cell lysis buffer will get functional and solubalbe GPCR in cell lysate supernatant. Wouid you mind introducing the more detail procedure to get functional membrane protein?
Thank you very mucn in advance.
Best
Zhiyuan
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