Configuration-specificMonoclonalAntibodyBasedGα13ActivationAssayKitCatalogNumber：8040120assaysProductDescriptionAstructurallydiverserepertoireofligands

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Configuration-specific Monoclonal Antibody Based

Gα13 Activation Assay Kit

Catalog Number：80401

20 assays

Product Description

A structurally diverse repertoire of ligands, from photons to large peptides, activates GPCRs to elicit

their physiological functions. Ligand-bound GPCRs, in turn, function as guanine nucleotide exchange

factors catalyzing the exchange of GDP bound on the Gα subunit with GTP in the presence of Gβγ,

causing the dissociation of the Gα subunit from the Gβγ dimer to form two functional units (Gα and

Gβγ). Both Gα and Gβγ subunits signal to various cellular signaling pathways. Based on the sequence

ａnd functional homologies, G proteins are grouped into four families: Gs, Gi, Gq, and G12 . As increasing

numbers of effectors and interacting proteins for these G proteins have been identified, the physiological

processes in which G proteins participate are multiplying.

Among the four subfamilies of G proteins, the function of G12/13 subfamily is less well understood. In

this family, there are two members, G12 and G13, that are expressed ubiquitously. Gα12 knockout mice

appeared normal. Gα13 knockout mice displayed embryonic lethality (~E9.5). The Gα13-/-mouse

embryos had defective vascular systems. G13 is also essential for receptor tyrosine kinase-induced

migration of fibroblast and endothelial cells.

NewEast Biosciences Gα13 Activation Assay Kit provides a simple and fast tool to monitor the

activation of Gα13. Each kit provides sufficient quantities to perform 20 assays.

Assay Principle

NewEast Biosciences Gα13 Activation Assay Kit bases on the configuration-specific anti-Gα13-GTP

monoclonal antibody to measure the active Gα13-GTP levels, either from cell extracts or from in vitro

GTPγS loading Gα13 activation assays. Briefly, anti-active Gα13 mouse monoclonal antibody will be

incubated with cell lysates containing Gα13-GTP. The bound active Gα13 will then be pulled down by

protein A/G agarose. The precipitated active Gα13 will be detected by immunoblot analysis using anti

Gα13 rabbit polyclonal antibody.

Kit Components

1. Anti-active Gα13, Mouse Monoclonal Antibody (Catalog No. 26902): One vial – 22 µL (1mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody

specifically recognizes Gα13-GTP from all vertebrates.

2. Protein A/G Agarose (Catalog No. 30301): One vial – 400 µL of 50% slurry. 3. 5X Assay/Lysis Buffer (Catalog No. 30303): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750mM NaCl, 5 mM EDTA, 5% Triton X-100.

4. Anti- Gα13, Rabbit Polyclonal Antibody (Catalog No. 21005): One vial – 22 µL (1 mg/ml) in

PBS, pH 7.4, contained 50% glycerol.

Storage

Store all kit components at 4oC until their expiration dates.

Materials Needed but Not Supplied

1. Stimulated and non-stimulated cell lysates

2. Protease inhibitors

3. 4 °C tube rocker or shaker

4. 1 M EDTA, pH8.0

5. 1 M MgCl2

6. 2X reducing SDS-PAGE sample buffer

7. Electrophoresis and immunoblotting systems

8. Immunoblotting wash buffer such 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. PVDF or nitrocellulose membrane

11. Secondary Antibody

12. ECL Detection Reagents

Reagent Preparation

? 1X Assay/Lysis Buffer: Mix the 5X Stock briefly and dilute to 1X in deionized water. Just prior to

usage, add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin.

Sample Preparation

Adherent Cells

1. Culture cells to approximately 80-90% confluence. Stimulate cells with activator or inhibitor as desired.

2. Aspirate the culture media and wash twice with ice-cold PBS.

3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer to the cells (0.5- 1 mL per 100 mm tissue culture plate).

4. Place the culture plates on ice for 10-20 minutes.

5. Detach the cells from the plates by scraping with a cell scraper.

6. Transfer the lysates to appropriate size tubes and place on ice.

7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this

occurs, lysates can be passed through a 27?-gauge syringe needle 3-4 times to shear the genomic DNA.

8. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

9. Collect the supernatant and store samples on ice for immediate use, or snap freeze and store at -70 °C for future use.

Suspension Cells

1. Culture cells and stimulate with activator or inhibitor as desired.

2. Perform a cell count, and then pellet the cells by centrifugation.

3. Aspirate the culture media and wash twice with ice-cold PBS.

4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer to the cell pellet

(0.5 – 1 mL per 1 x 10⁷cells).

5. Lyse the cells by repeated pipetting.

6. Transfer the lysates to appropriate size tubes and place on ice.

7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this

occurs, lysates can be passed through a 27?-gauge syringe needle 3-4 times to shear the genomic DNA.

8. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

9. Collect the supernatant and store samples on ice for immediate use, or snap freeze and store at -

70 °C for future use.

Assay Procedure

I. Active Gα13 Pull-Down Assay

1. Aliquot 0.5 – 1 mL of cell lysate to a microcentrifuge tube.

2. Adjust the volume of each sample to 1 mL with 1X Assay/Lysis Buffer.

3. Add 1 µl anti-active Gα13 monoclonal antibody to the tube.

4. Thoroughly resuspend the protein A/G Agarose bead slurry by vortexing or titurating.

5. Quickly add 20 µL of resuspended bead slurry to each tube.

6. Incubate the tubes at 4 °C for 1 hour with gentle agitation.

7. Pellet the beads by centrifugation for 1 min at 5,000 x g.

8. Aspirate and discard the supernatant, making sure not to disturb/remove the bead pellet.

9. Wash the bead 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.

10. After the last wash, pellet the beads and carefully remove all the supernatant.

11. Resuspend the bead pellet in 20 µL of 2X reducing SDS-PAGE sample buffer.

12. Boil each sample for 5 minutes.

13. Centrifuge each sample for 10 seconds at 5,000 x g.

II. Electrophoresis and Transfer

1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (8%). Also, it’s recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3).

2. Perform SDS-PAGE following the manufacturer’s instructions.

3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s

instructions.

III. Immunoblotting and Detection (all steps are at room temperature, with agitation)

1. Following the electroblotting step, immerse the PVDF membrane in 99% Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.

Note: If Nitrocellulose is used instead of PVDF, this step should be skipped.

2. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature

with constant agitation.Incubate the membrane with anti-Gα13 polyclonal antibody, freshly diluted 1:100 in 5% non-fat dry milk or 3% BSA/TBST, for 1-2 hr at room temperature with constant agitation or at 4^oC

overnight.

3. Wash the blotted membrane three times with TBST, 5 minutes each time.

4. Incubate the membrane with a secondary antibody (e.g. Goat Anti-Rabbit IgG, HRP-conjugate),

freshly diluted 1:1000 in 5% non-fat dry milk or 3% BSA/TBST, for 1 hr at room temperature

with constant agitation.

5. Wash the blotted membrane three times with TBST, 5 minutes each time.

6. Use the detection method of your choice such as ECL.

Example of Results

The following figure demonstrates typical results seen with NewEast Biosciences Gα13 Activation

Assay Kit. One should use the data below for reference only.

Gα13 activation assay. MEF cells were treated with (lane 2) or without (lane 1) LPA. Cell lysates

were incubated with an anti-active Gα13 monoclonal antibody (Cat. # 26902) (top panel). The

precipitated active Gα13 was immunoblotted with an anti- Gα13 rabbit polyclonal antibody (Cat #

21005). The bottom panel shows the Western blot with anti- Gα13 of the cell lysates used (5% of

that used in the top panel).

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	Anti-Gα ₁₃ Rabbit Polyclonal Antibody	100 μL	￥2600