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Gαi Pull-DownActivation Assay Kit
Cat.# 83001
Introduction
A.BackgroundA structurally diverse repertoireof ligands, from photons to large peptides, activates Gprotein-coupled receptors (GPCRs) to elicit their physiologicalfunctions. Ligand-bound GPCRs, in turn, function as guaninenucleotide exchange factors catalyzing the exchange of GDP bound onthe Gα subunit with GTP in the presence of Gβγ, causing thedissociation of the Gα subunit from the Gβγ dimer to form twofunctional units (Gα and Gβγ). Both Gα and Gβγ subunits signal tovarious cellular signaling pathways. Based on the sequence andfunctional homologies, G proteins are grouped into four families:Gs, Gi, Gq, and G12.
Gαi familyis the largest family of G proteins. They relay signals from manyGPCRs to regulate various biological functions. There were nodirect methods to measure the activation of Gαi Proteinsby receptors (until this assay kit). Most reports used one of thedownstream pathways, i.e. the inhibition of adenylyl cyclases, as areadout. Alternatively, sensitivity to pertussis toxin (PTX) wasused as an indicator of possible Gαi proteinsinvolved in a signaling pathway.
B.Assay PrincipleThe Gαi ActivationAssay Kit uses configuration-specific anti-Gαi-GTPMouse monoclonal antibody to measure Gαi-GTPlevels in cell extracts or in vitro GTPγS loadingGαi activationassays. Anti-Gαi-GTPmouse monoclonal antibody is first incubated with cell lysatescontaining Gαi-GTP.Next, the GTP-bound Gαi ispulled down by protein A/G agarose. Finally, the precipitatedGαi-GTPis detected through immunoblot analysis usinganti-Gαi mousemonoclonal antibody.
C.Kit Components1. Anti-Gαi-GTPMouse Monoclonal Antibody (Cat. # 26901): One vial – 35 µL (1mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibodyspecifically recognizes Gαi-GTPfrom all vertebrates.
2. Protein A/G Agarose (Cat. #30301): One vial – 600 µL of 50% slurry.
3. 5X Assay/Lysis Buffer (Cat. #30302): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750mM NaCl, 50mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Gαi Mousemonoclonal Antibody (Cat. # 26003): One vial – 50 µL (1mg/mL) inPBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. # 30303): Onevial – 50 µl at 10 mM, use 5 µL of GTPγS for GTP-labeling of0.5 mL of cell lysate.
6. 100X GDP (Cat. # 30304): Onevial – 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5 mLof cell lysate.
7. HRP-Goat Anti-Rabbit IgG (Cat.#29002): 50 µL (0.4 mg/mL) in PBS, pH 7.4, contained 50%glycerol.
D.Materials Needed but Not Supplied1. Stimulated and non-stimulatedcell lysates
2. Protease inhibitors
3. 4 °C tube rocker orshaker
4. 0.5 M EDTA at pH8.0
5. 1.0 M MgCl2
6. 2X reducing SDS-PAGE samplebuffer
7. Electrophoresis andimmunoblotting systems
8. Immunoblotting wash buffersuch as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05% Tween-20)
9. Immunoblotting blocking buffer(TBST containing 5% Non-fat Dry Milk or 3% BSA)
10. ECL DetectionReagents
E.Example ResultsThe following figure demonstratesexample results seen with the Gαi ActivationAssay Kit. For reference only.
Gαi ActivationAssay. A.CHO cells were transfected with wild-type Gαi1 (lanes1 and 2) or constitutively active Gαi1-Q204L(lane 3). Cell lysates were treated with GDP (lane 1) or GTPγS(lane 3). Lysates were then incubated with ananti-Gαi-GTPmonoclonal antibody (Cat. # 26901) (top panel). The precipitatedGαi-GTPwas immunoblotted with an anti-Gαi monoclonalantibody (Cat. # 26003). The bottom panel shows the Western blotwith anti-Gαi monoclonalantibody (Cat. # 26003) of the cell lysates. B. HEK293 cells stablyexpressing human m2 mAChR were treated with (lane 2) or without(lane 1) carbachol. Cell lysates were then incubated with ananti-active Gαi monoclonalantibody (Cat. No. 26901) (top panel). The precipitatedGαi-GTPwas immunoblotted with an anti-Gαi rabbitpolyclonal antibody (Cat. # 21006). The bottom panel shows theWestern blot with anti-tubulin of the celllysates.
AssayProcedure
A.Reagent Preparation1X Assay/Lysis Buffer: Mixthe 5X Stock (Cat. # 30301) briefly and dilute to 1X in deionizedwater. Just prior to usage, add protease inhibitors such as 1 mMPMSF, 10 µg/mL leupeptin, or 10 µg/mL aprotinin.
B.Sample PreparationAdherent Cells1. Culture cells (one 10-cmplate, ~107 cells)to approximately 80-90% confluence. Stimulate the cells withactivator or inhibitor as desired.
2. Aspirate the culture media andwash twice with ice-cold PBS.
3. Completely remove the finalPBS wash and add ice-cold 1X Assay/Lysis Buffer (See ReagentPreparation) to the cells (0.5-1 mL per 10 cm tissue cultureplate).
4. Place the culture plates onice for 10-20 minutes.
5. Detach the cells from theplates by scraping with a cell scraper.
6. Transfer the lysates toappropriate size tubes and place on ice.
7. If nuclear lysis occurs, thecell lysates may become viscous and difficult to pipette. If thisoccurs, lysates can be passed through a 27½-gauge syringe needle3-4 times to shear the genomic DNA.
8. Clear the lysates bycentrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant andstore the sample (~1-2 mg of total protein) on ice for immediateuse, or snap freeze and store at -70°C for future use.
Adherent Cells1. Culture cells and stimulatewith activator or inhibitor as desired.
2. Perform a cell count and thenpellet the cells through centrifugation.
3. Aspirate the culture media andwash twice with ice-cold PBS.
4. Completely remove the finalPBS wash and add ice-cold 1X Assay/Lysis Buffer (See ReagentPreparation) to the cell pellet (0.5-1 mL per 107 cells).
5. Lyse the cells by repeatedpipetting.
6. Transfer the lysates toappropriate size tubes and place them on ice.
9. Collect the supernatant andstore sample on ice for immediate use, or snap freeze and store at-70°C for future use.
C.In vitro GTPγS/GDP Protein for Positive and NegativecontrolsNote: In vivo stimulation of cells willactivate approximately 10% of the available Gαi,whereas in vitro GTPγS protein loading will activate nearly 90% ofGαi.
1. Aliquot 0.5 mL of cell extract(or 1 µg of purified Gαi protein)into two microcentrifuge tubes.
2. To each tube, add 20 µL of 0.5M EDTA (final concentration of 20 mM).
3. Positive control: add 5 µL of100 X GTPγS (Cat. # 30302) to the 1st tube
4. Negative control: add 5 µL of100 X GDP (Cat. # 30304) to the 2nd tube.
5. Incubate both tubes at 30°Cfor 30 minutes with agitation.
6. Stop loading by placing thetubes on ice and adding 32.5 µL of 1 M MgCl2 (finalconcentration of 60 mM).
D.Affinity Precipitation of Activated G Protein1. Aliquot 0.5-1 mL of celllysates (about 1 mg of total cellular protein) to a microcentrifugetube.
2. Adjust the volume to 1 mL with1X Assay/Lysis Buffer (See Reagent Preparation).
3. Add 1 µL anti-Gαi-GTPantibody (Cat. # 26901).
4. Prepare the protein A/GAgarose bead slurry (Cat. # 30301) by resuspending throughvertexing or titrating.
5. Quickly add 20 µL ofresuspended bead slurry to above tube.
6. Incubate the tube at 4°C for 1hour with gentle agitation.
7. Pellet the beads throughcentrifugation at 5,000 x g for 1 min.
8. Aspirate and discard thesupernatant (making sure not to disturb or remove the beadpellet.
9. Wash the beads 3 times with0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating eachtime.
10. After the third wash, pelletthe beads through centrifugation and carefully remove all thesupernatant.
11. Resuspend the bead pellet in20 µL of 2X reducing SDS- PAGE sample buffer.
12. Boil the sample for 5minutes.
13. Centrifuge it at 5,000 x gfor 10 seconds.
E.Western Blot Analysis1. Load 15 µL/well of pull-downsupernatant to a polyacrylamide gel (17%). It is recommended toinclude a pre-stained MW standard (as an indicator of a successfultransfer in step 3 below).
2. Perform SDS-PAGE following themanufacturer’s instructions.
3. Transfer the gel proteins to aPVDF or nitrocellulose membrane following the manufacturer’sinstructions.
Note: Steps 4-11 are at room temperaturewith agitation
4. Following electroblotting,immerse the PVDF membrane in Methanol for 15 seconds, andthen allow it to dry at room temperature for 5 minutes.
Note: If Nitrocellulose is used insteadof PVDF, step 4 Should be skipped.
5. Block the membrane with 5%non-fat dry milk or 3% BSA in TBST for 1 he at room temperaturewith constant agitation.
6. Wash the blotted membranethree times with TBST, 5 minutes each time.
7. Incubate the membrane withanti-Gαi MouseMonoclonal Antibody (Cat. # 26003), which has been freshly diluted1: 50~500 (depending on the amount of Gαi proteinsin your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2her at room temperature with constant agitation or at 4°Covernight.
8. Wash the blotted membranethree times with TBST, 5 minutes each time.
9. Incubate the membrane with asecondary antibody (Cat. # 29002), which has been freshly diluted1: 1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 he at roomtemperature with constant agitation.
10. Wash the blotted membranethree times with TBST, 5 minutes each time.
11. Use the detection method ofyour choice such as ECL.