Configuration-specificMonoclonalAntibodyBasedGαiActivationAssayKitCatalogNumber：8030120assaysProductDescriptionAstructurallydiverserepertoireofligands

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Configuration-specific Monoclonal Antibody Based

Gαi Activation Assay Kit

Catalog Number：80301

20 assays

Product Description

A structurally diverse repertoire of ligands, from photons to large peptides, activates G protein-coupled

receptors (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.

Gαi family is the largest family of G proteins. They relay signals from many GPCRs to regualte

various biological functions. There were no direct methods to measure the activation of Gαi proteins

by receptors (until this assay kit). Most reports used one of the downstream pathway, i.e. the

inhibition of adenylyl cyclases, as a readout. Alternatively, sensitivity to pertussis toxin (PTX) was

used as an indicator of possible Gαi proteins invovled in a signaling pathway.

NewEast Biosciences Gαi Activation Assay Kit provides a direct measurement of the activation of Gαi

proteins. This is a simple and fast tool to monitor the activation of Gαi. Each kit provides sufficient

quantities to perform 20 assays.

NewEast Biosciences Gαi Activation Assay Kit is based on the monoclonal antibody specifically

recognizing the active GTP-bound Gαi proteins. This monoclonal antibody has much lower affinity

towards the inactive Gαi proteins. Therefore, after activation by receptor signals, active GTP-bound

Gαi proteins could be immunoprecipitated by this monoclonal antibody and further quantified by

western blot with another anti- Gαi antibody.

Assay Principle

NewEast Biosciences Gαi Activation Assay Kit is an immunoprecipitation/western blot assay to

measure the levels of active GTP-bound Gαi proteins, either from cell extracts or from in vitro GTPγS

loaded Gαi proteins. Briefly, the anti-active Gαi monoclonal antibody will specifically bind to active

Gαi protein. This antibody/ Gαi complex will then be pulled down by protein A/G agarose. The

precipitated active Gαi proteins will be detected by immunoblots with another anti-Gαi antibody.

Kit Components

1. Anti-active Gαi, Mouse Monoclonal Antibody (Catalog No. 26901): One vial – 22 µL (1mg/mL) in PBS, pH 7.4, contained 50% glycerol. This antibody specifically recognizes GTP- Gαi 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 7.4,750
mMNaCl, 5 mM EDTA, 5% Triton X-100.

4. Anti-Gαi, Mouse Monoclonal Antibody (Catalog No. 26003): One vial – 22 µL(1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30302): One vial –100 µL at 10 mM, use 5 µL of GTPγS for

GTP-labeling of 0.5 mL of cell lysate.

6. 100 X GDP (Catalog No. 30304): One vial –100 µL at 100 mM, use 5 µL of GDP for

GDP-labeling of 0.5 mL of cell lysate.

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 MgCl2

5. 2X reducing SDS-PAGE sample buffer

6. Electrophoresis and immunoblotting systems

7. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05 %

Tween-20)

8. Immunoblotting blocking buffer (TBST containing 5 % Non-fat Dry Milk or 3 % BSA)

9. PVDF or nitrocellulose membrane

10. Secondary Antibody

11. 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 (one 10-cm plate, ~ 10⁷cells) 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 per10 cm 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. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

8. Collect the supernatant and store samples (~1-2 mg of total proteins) 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.

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.

In vitro GTPγS/GDP Protein Loading for positive and negative controls

Note: In vivo stimulation of cells with receptor ligands might activate ~10 % of the available Gαi

proteins, whereas in vitro GTPγS loading could activate ~50 % of the Gαi proteins that can be

activated.

1. Aliquot 0.5 mL of each cell extract to two microfuge tubes.

2. To each tube, add 5 µL of 1M MgCl2 (to 10 mM final concentration).

3. Add 5 µL of 100X GTPγS (to 100 µM, final concentration) to one tube (positive control).

4. Add 5 µL of 100X GDP (to 1 mM, final concentration) to the second tube (negative control).

5. Incubate the tubes at 30°C for 90 minutes with agitation.

Assay Procedure

I. Active Gαi 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 Gαi monoclonal antibody (Cat. No. 26901) to the tube.

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

5. 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 10 seconds at 12,000 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 12,000 x g.

II. Electrophoresis and Transfer

1. Load 20 µL/well of pull-down supernatant to a polyacrylamide gel. 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 as per the manufacturer’s instructions.

3. Transfer the gel proteins to a PVDF or nitrocellulose membrane as per 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-Gαi monoclonal antibody (Cat. No. 26003), freshly diluted

1:100 ~ 1000 in 5 % non-fat dry milk or 3 % BSA/TBST, for 1-2 hr at room temperature with

constant agitation.

Note: To conserve antibody, incubations should be performed in a plastic bag.

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-mouse IgG, HRP-conjugate),

freshly diluted 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.

Example of Results

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

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

Gαi activation assay. A. CHO cells were transfected with wild-type Gαi1 (lanes 1 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 an anti-active Gαi monoclonal antibody (Cat. No. 26901) (top panel). The precipitated active Gαi was immunoblotted with an anti- Gαi monoclonal antibody (Cat. No. 26003). The bottom panel shows the Western blot with anti- Gαi monoclonal antibody (Cat.No.26003) of the cell lysates. B. HEK293 cells stably expressing human m2 mAChR were treated with(lane 2) or without (lane 1) carbachol. Cell lysates were then incubated with an anti-active Gαi monoclonal antibody (Cat. No. 26901) (top panel). The precipitated active Gαi was immunoblotted with an anti- Gαi rabbit polyclonal antibody (Cat. No. 21006). The bottom panel shows the Western blot with anti-tubulin of the cell lysates.

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