Protocol to characterize Gi/o and Gs protein-coupled receptors in transiently transfected cells using ELISA and cAMP measurements

. 2023 Feb 14;4(1):102120. doi: 10.1016/j.xpro.2023.102120

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Anti-HA-Peroxidase, High Affinity from rat IgG1 monoclonal (1:1000)	Sigma-Aldrich	12013819001RRID: AB_390917
Anti-FLAG M2 antibody produced in mouse (1:100)	Sigma-Aldrich	F3165RRID: AB_259529

Oligonucleotides

GPCR open reading frames generated from genomic DNA or purchased as synthetic DNA	See references¹^,²^,³^,⁴^,⁵^,⁶^,⁷^,⁸	N/A
HA-adapter primer-S (human GPR84)	Schulze et al.¹	acgtccccgactacgccTGGAACAGCTCTGACGCC
FLAG-adapter primer-AS (human GPR84)	Schulze et al.¹	TCActtatcgtcatcgtccttatagtcATGGAGCCTATGGAA
HA-universal primer EcoRI-S	Schulze et al.¹	cgcGAATTCgccaccATGtacccctacgacgtccccgactacg
FLAG-universal primer SpeI-AS	Schulze et al.¹	cgccgcACTAGTTCActtatcgtcatcgtccttatagtc

Chemicals, peptides, and recombinant proteins

10× HBSS	Thermo Fisher Scientific	14065056
1× D-PBS	Thermo Fisher Scientific	14190144
3-Isobutyl-1-methylxanthine (IBMX)	Sigma-Aldrich	I5879
Bovine serum albumin (BSA)	Sigma-Aldrich	A3294
Citric acid	Sigma-Aldrich	251275
Deoxynucleotide (dNTP) Solution Set	NEB	N0446
Dulbecco’s Modified Eagle Medium (DMEM)	Thermo Fisher Scientific	41966029
Dulbecco’s Modified Eagle Medium (DMEM)/ F-12	Thermo Fisher Scientific	21041025
Dithiothreitol (DTT)	Sigma-Aldrich	DTT-RO
UltraPure 0.5 M EDTA, pH 8	Thermo Fisher Scientific	15575020
Fetal bovine serum (FBS)	Thermo Fisher Scientific	10270106
Formaldehyde	Sigma-Aldrich	F8775
Forskolin	Sigma-Aldrich	F3917
H₂O₂	Sigma-Aldrich	1.08600
HCl	Sigma-Aldrich	258148
HEPES (1 M)	Thermo Fisher Scientific	15630056
Lipofectamine 2000	Thermo Fisher Scientific	11668019
Na₂HPO₄ · 2 H₂O	Sigma-Aldrich	S9763
Na₂SO₃	Sigma-Aldrich	S0505
Nonidet P40 (substitute) (NP-40)	Santa Cruz	sc-29102
o-phenylenediamine	Sigma-Aldrich	P23938
Opti-MEM	Thermo Fisher Scientific	31985070
Penicillin-Streptomycin (5.000 U/mL)	Thermo Fisher Scientific	15140122
Poly-L-lysine solution (0.1% (w/v) in H₂O)	Sigma-Aldrich	P8920
Q5 High-Fidelity DNA Polymerase	NEB	M0491
Sodium-Deoxycholate	Sigma-Aldrich	30970
Sodium tetraborate decahydrate	Sigma-Aldrich	S9640
Triton X-100	Sigma-Aldrich	T8787
Tris/HCl	Sigma-Aldrich	10812846001
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200056
Tween 20	Sigma-Aldrich	P1379

Critical commercial assays

AlphaScreen cAMP assay kit	PerkinElmer Life Sciences	6760635M

Experimental models: Cell lines

CHO-K1 (passage #3 to max. #40)	ATCC	CCL-61
HEK293-T (passage #3 to max #40)	ATCC	CRL-3216

Recombinant DNA

N-HA-GPCR-FLAG-C-tagged receptor in pcDps	See references¹^,²^,³^,⁴^,⁵^,⁶^,⁷^,⁸	N/A

Software and algorithms

GraphPad Prism	GraphPad Software	https://www.graphpad.com/scientific-software/prism/
Bio-Render	Bio-Render	https://biorender.com/

Other

Maxi-Sorp flat bottom 96-well plates	Thermo Fisher Scientific	44-2404-21
OptiPlate-384, White Opaque 384-well Microplate	PerkinElmer Life Sciences	6007290
25 cm² cell culture flask	Greiner Bio-One	690175
75 cm² cell culture flask	Greiner Bio-One	658175
6-well plate	Greiner Bio-One	657160
48-well plate	Greiner Bio-One	655180
96-well plate	Greiner Bio-One	677165
Multimode multilabel plate reader able to detect PerkinElmer Alpha Technology	PerkinElmer Life Sciences	EnVision 2104 multilabel plate reader with barcode #444 mirror (cat#2101-4010), barcode #244 AlphaScreen 570/100 filter (cat #2100-5710) equipped with laser (excitation: 680 nm) by default)
Plate reader to measure absorbance at 492 nm and 620 nm	Tecan	Tecan-Reader Infinite M Nano
Orbital shaker	Heidolph	Heidolph Titramax 1000
Shaker with 37°C incubator hood	Edmund Bühler	Edmund Bühler shaker SM30 with incubator hood TH30
Overhead shaker	Heidolph	Heidolph Reax 2
Multi-dispensing multi-channel pipettes	Gilson/Brandt/Thermo	Pipetman G Multichannel P12x200G (12 channel) Tranferpette S-12, (0.5–10 μL) Finnpipette Novus (16 channel)
Aspiration system	Biovac	Biovac 106 with 2 l glass bottle