Mar 12, 2020
Monitoring cell-surface expression of GPCR by ELISA
- Elie Besserer-Offroy1,
- Rebecca L Brouillette2,
- Jean-Michel Longpré2,
- Philippe Sarret2
- 1Inserm U1086 - Anticipe;
- 2Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke
- The Besserer LabTech. support email: protocol_support@bessererlab.org
Protocol Citation: Elie Besserer-Offroy, Rebecca L Brouillette, Jean-Michel Longpré, Philippe Sarret 2020. Monitoring cell-surface expression of GPCR by ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.zfef3je
Manuscript citation:
Gusach A, Luginina A, Marin E, Brouillette RL, Besserer-Offroy É, Longpré JM, Ishchenko A, Popov P, Patel N, Fujimoto T, Maruyama T, B Stauch, Ergasheva M, Romanovskaia D, Stepko A, Kovalev K, Shevtsov M, Gordeliy V, Han GW, Katritch V, Borshchevskiy V, Sarret P, Mishin A, Cherezov V. Structural basis of ligand selectivity and disease mutations in cysteinyl leukotriene receptors. Nature Communications. 2019; (10):5573. PMID: 31811124.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 22, 2019
Last Modified: March 12, 2020
Protocol Integer ID: 21702
Keywords: G Protein-Coupled Receptor, Seven Transmembrane Receptor, Cell Surface Expression, ELISA, HA-tag, HRP-linked anti-HA,
Abstract
Quantifying cell surface expression of G Protein-Coupled Receptors (GPCRs) can be exteremly important for the expression of mutant receptors. Herein we report a useful Enzyme-Linked Immunosorbent Assay (ELISA) for the cell-surface detection of a HA-tagged version of a GPCR.
Materials
MATERIALS
NESTLE CARNATION Instant Non-fat Dry Milk
Poly-l-lysine, 0.1% (wt/vol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8920
Falcon® 24-Well Flat-Bottom Plate, Tissue Culture-Treated 50 Plates
STEMCELL Technologies Inc.Catalog #38021
HEK293ATCCCatalog #CRL-1573
Lipofectamine 3000Thermo Fisher ScientificCatalog #L3000015
Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985062
Sterile WaterWisent BioproductsCatalog #809-115-CL
PBS 1XWisent BioproductsCatalog #311-011-CL
Trypsin 0.25% / EDTA 2.21 mM in HBSSWisent BioproductsCatalog #325-043-EL
SafeSeal tube 5mLSarstedtCatalog #72.701
Formaldehyde Reagent GradeBioshopCatalog #FOR201
3 3′5 5′-Tetramethylbenzidine Liquid Substrate Supersensitive for ELISAMerck MilliporeSigma (Sigma-Aldrich)Catalog #T4444-100ML
Anti-HA-Peroxidase High AffinityMerck MilliporeSigma (Sigma-Aldrich)Catalog #12013819001
FBS (Fetal Bovine Serum) Premium Quality Endotoxin Wisent BioproductsCatalog #080-150
DMEM 4.5g/L glucose with L-glutamine sodium pyruvate and phenol redWisent BioproductsCatalog #319-005-CL
HEPES 1M Free acidWisent BioproductsCatalog #330-050-EL
Penicillin (5000IU) / Streptomycin (5000μg/mL) sterile filtered for cell cultureWisent BioproductsCatalog #450-200-EL
Endotoxin-free purified plasmidic DNA encoding for HA-tagged GPCRs
Tris-Buffered Saline (TBS, containing 20mM Tris–HCl pH 7.5 and 150mM NaCl)
Day 1 - Cell Culture & Transfections
Day 1 - Cell Culture & Transfections
Coat 24-well plates with Poly-L-Lysine (this need to be done in a biological safety cabinet to ensure sterility).
Add 300 µL of 0.1 mg/mL Poly-L-Lysine solution in each well of the 24-well plate and incubate 00:10:00 at Room temperature .
This can be done using a 300µL multichannel pipet fitted with 4 tips
Remove the Poly-L-Lysine solution (this solution can be re-used up to 4 times to coat cell culture plasticware).
This can be done using a 300µL multichannel pipet fitted with 4 tips
Rinse the wells twice with 300 µL of sterile water.
Let dry the 24-well plate without lid under the biological safety cabinet for 00:20:00 before seeding cells.
Note
Poly-L-Lysine-coated plates can be stored for several weeks at Room temperature befor use.
Prepare transfections of plasmids encoding HA tagged-GPCRs (this need to be done in a biological safety cabinet to ensure sterility).
Note
In the case you want to compare expression of a mutant receptor, positive and negative controls are needed to normalize the results. The positive control should be the wild-type receptor and the negative control should be cells transfected with the empy vector (MOCK cells).
For 3 well of the 24-well plate:
Add 300 µL of Opti-MEM into a sterile 5mL tube.
Add 1.5 µg of plasmidic DNA encoding for the desired HA tagged-GPCR to the tube containing Opti-MEM and mix.
Add 3 µL of P3000 Reagent to the tube and mix.
Add 2.25 µL of Lipofectamine 3000 to the tube, mix, and incubate at Room temperature for 00:15:00 .
Prepare HEK293 cells for transfection (this need to be done in a biological safety cabinet to ensure sterility).
Note
Ideally, cells were seeded at a density of 3 million cells per 10cm-pretri dish 48h before transfection to ensure a high transfection rate.
Remove culture media and rinse cells with PBS.
Add 1 mL of 0.25% Trypsin to a 10-cm petri dish and incubate for 00:02:00 at 37 °C .
Add 5 mL of complete DMEM (10% FBS, 20mM HEPES, Penicilin/Streptomycin) to the petri dish and dissociate cells by pipeting up and down.
Count cells using an automated cell counter or a hemacytometer.
Equipment
MOXI Z Mini
NAME
Automated Cell Counter
TYPE
Orflo
BRAND
MXZ001
SKU
LINK
Adjust cell concentration to 150,000 cells/mL.
Add 2 mL of the cell suspension at 150,000 cells/mL to the 5mL tube containing the plasmidic DNA and mix gently.
Dispense 450 µL of the mix of cell and plasmidic DNA to the desired wells of the Poly-L-Lysine-coated 24-well
plate.
Incubate at 37 °C in humidified chamber at 5% CO2 for 48:00:00 .
Day 3 - ELISA
Day 3 - ELISA
Detection of cell surface expression by ELISA (this part of the protocol can be done on the wet bench).
Note
Buffer and reagents in this part of the protocol can be dispensed to the 24-well plate using combitips and a single channel repeater.
Equipment
M4 Repeater
NAME
Multidispense pipet
TYPE
Eppendorf
BRAND
4982000322
SKU
LINK
Remove cell culture media and wash each well with 400 µL of PBS.
Fix cells using 400 µL of 3.7 Mass Percent formaldehyde in TBS for 00:05:00 at Room temperature .
Rinse cells three times with 500 µL of TBS.
Block non-specific sites using 400 µL of 3 Mass Percent non-fat dry milk disolved in TBS for 00:30:00 under gentle orbital agitation.
Note
In case non-fat dry milk is not suitable for blocking non-specific sites, a solution of 1 Mass Percent of Bovine Serum Albumine in TBS can be used. The same solution must also be used for the incubation of the antibody.
Remove blocking solution and add 250 µL of 1/1000 dilution of HRP-linked anti-HA antiboby diluted in 3 Mass Percent non-fat dry milk in TBS for 03:00:00 under gentle orbital agitation.
Note
This incubation step can also be done Overnight at 4 °C under gentle orbital agitation.
Remove antibody solution and wash each well three times with 500 µL of TBS.
Add 250 µL of Room temperature 3, 3′,5 ,5′-Tetramethylbenzidine (TMB) Liquid Substrate, Supersensitive, for ELISA and incubate under gentle orbital agitation for 2 to 15 min (until the color of your positive control turn intense blue).
Stop the TMB reaction by adding 250 µL of 2 Molarity (M) Hydroclhoric Acid (HCl).
Transfer 100 µL of the colorimetric reaction to a flat-bottom transparent 96-well plate.
Read the absorbance at 450nm using a multimode plate reader.
Equipment
Mithras2 LB943
NAME
Multimode plate reader
TYPE
Berthold
BRAND
LB943
SKU
Results analysis
Results analysis
As this quantification of cell surface expression is a semi-quantitative method it should not be presented as raw OD450nm values but rather as a percentage of expression compared to the positive control (or wild-type receptor).
To normalize the results, average the OD450nm of the positive control and the OD450nm of the negative control and apply the following formula:
Normalization formula
y = normalized value
x = OD450nm value of the sample
xmin = mean OD450nm value of the negative control
xmax = mean OD450nm value of the positive control