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We have designed stringent quality control criteria to qualify all of our PathHunter® oGPCR cell lines. These criteria ensure that our oGPCR cell lines are viable for the duration of the assay, express the indicated oGPCR target at an abundant level and, more importantly, that oGPCR is capable of interacting with β-Arrestin.
1. Confirm Correct Molecular Weight
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Figure 1. Expression analysis of oGPCR
Cell lysates were analyzed using the PathHunter® EAstern Blot Detection Kit (P/N 93-0053). This convenient and antibody-free procedure is virtually identical to a Western blot, except that instead of using an antibody specific to the GPCR, EA reagent is used to universally detect any ProLink-tagged species. In this system, exogenously added EA is able to complement the immobilized ProLink-tagged GPCR, forming an active β-gal enzyme, capable of hydrolyzing substrate and generating a chemiluminescent signal.
Cell lysates were analyzed using the PathHunter® EAstern Blot Detection Kit (P/N 93-0053). This convenient and antibody-free procedure is virtually identical to a Western blot, except that instead of using an antibody specific to the GPCR, EA reagent is used to universally detect any ProLink-tagged species. In this system, exogenously added EA is able to complement the immobilized ProLink-tagged GPCR, forming an active β-gal enzyme, capable of hydrolyzing substrate and generating a chemiluminescent signal.
2. Quantify Basal Interaction Between the oGPCR and β-Arrestin
(ProLink Detection Kit, Cat. #92-0006)
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Figure 2. Basal interaction between oGPCR and β-Arrestin
When compared to the parental cell line, which only expresses the EA-Arrestin fusion protein, the GPR6 expressing cell line exhibits a 19x increase in basal interaction between GPR6 and β-Arrestin (982 vs. 53). In the presence of exogenous EA reagent, the fold increase is even greater, demonstrating abundant expression of GPR6 and an even greater potential for increased interaction if the GPCR were activated by a ligand.
When compared to the parental cell line, which only expresses the EA-Arrestin fusion protein, the GPR6 expressing cell line exhibits a 19x increase in basal interaction between GPR6 and β-Arrestin (982 vs. 53). In the presence of exogenous EA reagent, the fold increase is even greater, demonstrating abundant expression of GPR6 and an even greater potential for increased interaction if the GPCR were activated by a ligand.
3. Determine Viability of PathHunter® oGPCR cells
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Figure 3. Viability of PathHunter® oGPCR cells
PathHunter® oGPCR cells were plated for 48 hours in Cell Plating reagent to confirm viability and attachment.
PathHunter® oGPCR cells were plated for 48 hours in Cell Plating reagent to confirm viability and attachment.
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