武汉费斯生物科技有限公司的活性G蛋白检测试剂盒
是基于特异性识别与 GTP 结合的,而不认识 GDP 结合的GTP酶的单克隆抗体。
鉴于单克隆抗体对GTP酶的高亲和力，可以在短时间内进行活化测定。该试剂盒提供了具有高度灵敏性和可靠性。
Gαz Pull-Down Activation AssayKit
Cat. #81001
Introduction
A.BackgroundA structurally diverserepertoire of 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 family (includingGαz) is the largest family of G proteins. They relay signals frommany GPCRs to regulate various biological functions. There were nodirect methods to measure the activation of Gαz proteins byreceptors (until this assay kit). Most reports used one of thedownstream pathways, i.e. the inhibition of adenylyl cyclases, as areadout.
Gαz Activation AssayKit is based on the monoclonal antibody specifically recognizingthe active GTP-bound Gαz proteins. This monoclonal antibodyhas much lower affinity towards the inactive Gαz proteins.Therefore, after activation by receptor signals, active GTP-boundGαz proteins could be immunoprecipitated by this monoclonalantibody and further quantified by western blot with anotheranti-Gαz antibody.
B. AssayPrincipleThe Gαz ActivationAssay Kit uses configuration-specific anti-Gαz-GTP Mouse monoclonalantibody to measure Gαz-GTP levels in cell extracts or in vitroGTPγS loading Gαz activation assays. Anti-Gαz-GTP mousemonoclonal antibody is first incubated with cell lysates containingGαz-GTP. Next, the GTP-bound Gαz is pulled down by protein A/Gagarose. Finally, the precipitated Gαz-GTP is detected throughimmunoblot analysis using anti-Gαz mouse monoclonalantibody.
C. KitComponents1. Anti-Gαz-GTP MouseMonoclonal Antibody (Cat. # 26908): One vial – 35 µL (1 mg/ml) inPBS, pH 7.4, containing 50% glycerol. This antibody specificallyrecognizes Gαz-GTP from 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, 750mMNaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Gαz Mousemonoclonal Antibody (Cat. # 26011): One vial – 50 µL (1mg/mL) inPBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. #30303): One vial – 50 µl at 10 mM, use 5 µL of GTPγS for GTP-labeling of 0.5 mL of cell lysate.
6. 100X GDP (Cat. # 30304):One vial – 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5mL of 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 NotSupplied1. Stimulated andnon-stimulated cell lysates
2. Proteaseinhibitors
3. 4°C tube rocker orshaker
4. 0.5 M EDTA at pH8.0
5. 1.0 MMgCl2
6. 2X reducing SDS-PAGEsample buffer
7. Electrophoresis andimmunoblotting systems
8. Immunoblotting washbuffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl,0.05% Tween-20)
9. Immunoblotting blockingbuffer (TBST containing 5% Non-fat Dry Milk or 3%BSA)
10. ECL DetectionReagents
E. ExampleResultsThe following figuredemonstrates example results seen with the Gαz ActivationAssay Kit. For reference only.
Gαz ActivationAssay.Purified Gαz proteins were loaded as a control (lanes 1)or immunoprecipitated after treated with GDP (lane 2) or GTPγS(lane 3). Immunoprecipitation was done with the anti-Gαz-GTPmonoclonal antibody (Cat. # 26908). Immunoblot was with an anti-Gαz polyclonal antibody (Cat. # 26011).
AssayProcedure
A. ReagentPreparation1X Assay/LysisBuffer: Mix the 5X Stock (Cat. # 30301) briefly and dilute to1X in deionized water. Just prior to usage, add protease inhibitorssuch as 1 mM PMSF, 10 µg/mL leupeptin, or 10 µg/mLaprotinin.
B. SamplePreparationAdherentCells1. Culture cells (one 10-cmplate, ~107 cells) to approximately 80-90% confluence.Stimulate the cells with activator or inhibitor asdesired.
2. Aspirate the culturemedia and wash twice with ice-cold PBS.
3. Completely remove thefinal PBS 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 plateson ice 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,the cell lysates may become viscous and difficult to pipette. Ifthis occurs, lysates can be passed through a 27½-gauge syringeneedle 3-4 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 supernatantand store the sample (~1-2 mg of total protein) on ice forimmediate use, or snap freeze and store at -70°C for futureuse.
AdherentCells1. Culture cells andstimulate with activator or inhibitor as desired.
2. Perform a cell count andthen pellet the cells through centrifugation.
3. Aspirate the culturemedia and wash twice with ice-cold PBS.
4. Completely remove thefinal PBS wash and add ice-cold 1X Assay/Lysis Buffer (See ReagentPreparation) to the cell pellet (0.5-1 mL per107 cells).
5. Lyse the cells byrepeated pipetting.
6. Transfer the lysates toappropriate size tubes and place them on ice.
9. Collect the supernatantand store sample on ice for immediate use, or snap freeze and storeat -70°C for future use.
C. In vitro GTPγS/GDPProtein for Positive and Negative controlsNote: In vivo stimulation ofcells will activate approximately 10% of the available Gαz, whereasin vitro GTPγS protein loading will activate nearly 90% ofGαz.
1. Aliquot 0.5 mL of cellextract (or 1 µg of purified Gαz protein) into twomicrocentrifuge tubes.
2. To each tube, add 20 µLof 0.5 M EDTA (final concentration of 20 mM).
3. Positive control: add 5µL of 100 X GTPγS (Cat. # 30302) to the 1st tube
4. Negative control: add 5µL of 100 X GDP (Cat. # 30304) to the 2nd tube.
5. Incubate both tubes at30°C for 30 minutes with agitation.
6. Stop loading by placingthe tubes on ice and adding 32.5 µL of 1 M MgCl2 (finalconcentration of 60 mM).
D. Affinity Precipitationof 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 mLwith 1X Assay/Lysis Buffer (See ReagentPreparation).
3. Add 1 µL anti-Gαz-GTPantibody (Cat. # 26908).
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°Cfor 1 hour 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 timeswith 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspiratingeach time.
10. After the third wash,pellet the beads through centrifugation and carefully remove allthe supernatant.
11. Resuspend the beadpellet in 20 µL of 2X reducing SDS- PAGE samplebuffer.
12. Boil the sample for 5minutes.
13. Centrifuge it at 5,000 xg for 10 seconds.
E. Western BlotAnalysis1. Load 15 µL/well ofpull-down supernatant to a polyacrylamide gel (17%). It isrecommended to include a pre-stained MW standard (as an indicatorof a successful transfer in step 3 below).
2. Perform SDS-PAGEfollowing the manufacturer’s instructions.
3. Transfer the gel proteinsto a PVDF or nitrocellulose membrane following the manufacturer’sinstructions.
Note: Steps 4-11 are at roomtemperature with agitation
4. Followingelectroblotting, immerse the PVDF membrane in **** Methanol for 15seconds, and then allow it to dry at room temperature for 5minutes.
Note: If Nitrocellulose isused instead of PVDF, step 4 Should be skipped.
5. Block the membrane with5% 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 membranewith anti-Gαz Mouse Monoclonal Antibody (Cat. # 26011), whichhas been freshly diluted 1: 50~500 (depending on the amount ofGαz proteins in your sample) in 5% non-fat dry milk or 3% BSAin TBST, for 1-2 her at room temperature with constant agitation orat 4°C overnight.
8. Wash the blotted membranethree times with TBST, 5 minutes each time.
9. Incubate the membranewith a secondary antibody (Cat. # 29002), which has been freshlydiluted 1: 1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 heat room temperature with constant agitation.
10. Wash the blottedmembrane three times with TBST, 5 minutes eachtime.
11. Use the detection methodof your choice such as ECL.