Gαo Pull-Down Activation Assay Kit丨美国NewEastBio

我公司将向你提供以下的的三种抗体或者试剂盒: (1) 仅识别 GTP酶的活性构型的产品,它可以让你能够量化GTP酶在细胞中的活性和分布。(2) 识别突变 Oncogene蛋白, 但不认识相应野生型的抗体。 (3) 对cAMP 和 cGMP具有超亲和力（无需乙酰化）ELISA检测试剂盒。这些产品被将近一千篇同行评议的文章所引用。

Gαo Pull-DownActivation Assay Kit

Cat.# 80901

Introduction

A.Background

A 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 family(including Gαo)is 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 Gαo proteinsby receptors (until this assay kit). Most reports used one of thedownstream pathways, i.e. the inhibition of adenylyl cyclases, as areadout.

B.Assay Principle

NewEast Biosciences GαoActivationAssay Kit uses configuration-specific anti-Gαo-GTPMouse monoclonal antibody to measure Gαo-GTPlevels in cell extracts or in vitro GTPγS loadingGαoactivationassays. Anti-Gαo-GTPmouse monoclonal antibody is first incubated with cell lysatescontaining Gαo-GTP.Next, the GTP-bound Gαoispulled down by protein A/G agarose. Finally, the precipitatedGαo-GTPis detected through immunoblot analysis usinganti-Gαomousemonoclonal antibody.

C.Kit Components

1. Anti-Gαo-GTPMouse Monoclonal Antibody (Cat. # 26907): One vial – 35 µL (1mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibodyspecifically recognizes Gαo-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-GαoMousemonoclonal Antibody (Cat. # 21015): 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 Supplied

1. 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 Results

The following figure demonstratesexample results seen with the GαoActivationAssay Kit. For reference only.

Gαo ActivationAssay. PurifiedGαo proteinswere loaded as a control (lanes 1) or immunoprecipitated aftertreated with GDP (lane 2) or GTPγS (lane 3). Immunoprecipitationwas done with the anti-Gαo-GTPmonoclonal antibody (Cat. # 26907). Immunoblot was with ananti-Gαo polyclonalantibody (Cat. # 21015).

AssayProcedure

A.Reagent Preparation

1X 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 Cells

1. 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 Cells

1. 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 Negativecontrols

Note: In vivo stimulation of cells willactivate approximately 10% of the available Ar13, whereas in vitroGTPγS protein loading will activate nearly 90% ofAr13.

1. Aliquot 0.5 mL of cell extract(or 1 µg of purified Ar13 protein) into two microcentrifugetubes.

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 Protein

1. 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-Ar13-GTPantibody (Cat. # 26907).

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 Analysis

1. 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, and thenallow 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-Ar13 Mouse Monoclonal Antibody (Cat. # 21015), which has beenfreshly diluted 1: 50~500 (depending on the amount of Ar13 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.