Configuration-specific Monoclonal Antibody Based

Gαo Activation Assay Kit

Catalog Number：CR-W80901

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 (including Gαo) 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

Gαo 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.

   Bioyears Biosciences Gαo Activation Assay Kit provides a direct measurement of the activation of Gαo

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

quantities to perform 20 assays.

   Bioyears Biosciences Gαo Activation Assay Kit is based on the monoclonal antibody specifically

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

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

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

western blot with another anti Gαo antibody.

Assay Principle

   Bioyearst Biosciences Gαo Activation Assay Kit is an immunoprecipitation/western blot assay to

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

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

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

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

Kit Components

1. Anti active Gαo, Mouse Monoclonal Antibody (Catalog No. CR-M26907): One vial – 22 µL (1mg/mL) in

   PBS, pH 7.4, contained 50% glycerol. This antibody specifically recognizes GTP- Gαo from all ertebrates.

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 mM

   NaCl, 5 mM EDTA, 5% Triton X-100.

4. Anti Gα0, Rabbit Polyclonal Antibody (Catalog No. CR-R21015): 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. For long-term usage (more than 6 months),

please keep the antibodies at -20oC.

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