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i Pull-Down Activation Assay Kit

i Pull-Down Activation Assay Kit
5/5

$737.00

Cat.#:  80301

   Size:   30 Assays

In Stock

          Product Description          

i Pull-Down Activation Assay Kit

Cat. # 83001

Introduction

A. Background
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.
i family is the largest family of G proteins. They relay signals from many GPCRs to regulate 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 pathways, 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 involved in a signaling pathway.
B. Assay Principle

The Gαi Activation Assay Kit uses configuration-specific anti-Gαi-GTP Mouse monoclonal antibody to measure Gαi-GTP levels in cell extracts or in vitro GTPγS loading Gαi activation assays. Anti-Gαi-GTP mouse monoclonal antibody is first incubated with cell lysates containing Gαi-GTP. Next, the GTP-bound Gαi is pulled down by protein A/G agarose. Finally, the precipitated Gαi-GTP is detected through immunoblot analysis using anti-Gαi mouse monoclonal antibody.

C. Kit Components
1. Anti-Gαi-GTP Mouse Monoclonal Antibody (Cat. # 26901): 30 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Gαi-GTP from all vertebrates.
2. Protein A/G Agarose (Cat. # 30301): 600 µL of 50% slurry.
3. 5X Assay/Lysis Buffer (Cat. # 30302): 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Gαi Mouse monoclonal Antibody (Cat. # 26003): 50 µL (1mg/mL) in PBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. # 30303): 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): 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5 mL 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 Not Supplied
1. Stimulated and non-stimulated cell lysates
2. Protease inhibitors
3. 4 °C tube rocker or shaker
4. 0.5 M EDTA at pH 8.0
5. 1.0 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. ECL Detection Reagents
E. Example Results
The following figure demonstrates example results seen with the Gαi Activation Assay Kit. For reference only.

<img class="aligncenter" title="Gαi Activation Assay Kit” src=”/wp-content/uploads/ne/181418103620.png” alt=”Gαi Activation Assay Kit” data-mce-src=”/wp-content/uploads/ne/181418103620.png”>
i 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-Gαi-GTP monoclonal antibody (Cat. # 26901) (top panel). The precipitated Gαi-GTP was immunoblotted with an anti-Gαi monoclonal antibody (Cat. # 26003). The bottom panel shows the Western blot with anti-Gαi monoclonal antibody (Cat. # 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 Gαi-GTP was immunoblotted with an anti-Gαi rabbit polyclonal antibody (Cat. # 21006). The bottom panel shows the Western blot with anti-tubulin of the cell lysates.

Assay Procedure

A. Reagent Preparation

1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30302) briefly and dilute with deionized water to make 1X buffer. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin.

B. Sample Preparation
Adherent Cells
1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the 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 (See Reagent Preparation) to the cells (0.5-1 mL per 10 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 viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store the sample (~1-2 mg of total protein) 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 through 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 (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place them on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store 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 Negative controls
Note: In vivo stimulation of cells will activate approximately 10% of the available Gαi, whereas in vitro GTPγS protein loading will activate nearly 90% of Gαi.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Gαi protein) into two microcentrifuge tubes.
2. To each tube, add 20 µL of 0.5 M EDTA (final concentration of 20 mM).
3. Add 5 µL of 100 X GTPγS (Cat. # 30303) to the first tube as a positive control.
4. Add 5 µL of 100 X GDP (Cat. # 30304) to the second tube as a negative control.
5. Incubate both tubes at 30°C for 30 minutes with agitation.
6. Stop loading by placing the tubes on ice and adding 32.5 µL of 1 M MgCl2 (final concentration of 60 mM).
D. Affinity Precipitation of Activated G Protein
1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of total cellular protein) to a microcentrifuge tube.
2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (See Reagent Preparation).
3. Add 1 µL anti-Gαi-GTP antibody (Cat. # 26901).
4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) by resuspending through vertexing or titrating.
5. Quickly add 20 µL of resuspended bead slurry to above tube.
6. Incubate the tube at 4°C for 1 hour with gentle agitation.
7. Pellet the beads through centrifugation at 5,000 x g for 1 min.
8. Aspirate and discard the supernatant (making sure not to disturb or remove the bead pellet).
9. Wash the beads 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.
10. After the third wash, pellet the beads through centrifugation and carefully remove all the supernatant.
11. Resuspend the bead pellet in 20 µL of 2X reducing SDS- PAGE sample buffer.
12. Boil the sample for 5 minutes.
13. Centrifuge it at 5,000 x g for 10 seconds.
E. Western Blot Analysis
1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (17%). It is recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3 below).
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.
Note: Steps 4-11 are at room temperature with agitation
4. Following electroblotting, immerse the PVDF membrane in 100% Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.
Note: If Nitrocellulose is used instead of 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 temperature with constant agitation.
6. Wash the blotted membrane three times with TBST, 5 minutes each time.
7. Incubate the membrane with anti-Gαi Mouse Monoclonal Antibody (Cat. # 26003), which has been freshly diluted 1:50~500 (depending on the amount of Gαi proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 her at room temperature with constant agitation or at 4°C overnight.
8. Wash the blotted membrane three times with TBST, 5 minutes each time.
9. Incubate the membrane with a secondary antibody (Cat. # 29002), which has been freshly diluted 1:1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 he at room temperature with constant agitation.
10. Wash the blotted membrane three times with TBST, 5 minutes each time.
11. Use the detection method of your choice such as ECL.
          Publications          
01. GPER mediates estrogen cardioprotection against epinephrine-induced stress
J Endocrinol. 2021  PMID: 33847279
02. Analgesic Efficacy of α2 Adrenergic Receptor Agonists Depends on the Chronic State of Neuropathic Pain: Role of Regulator of G Protein Signaling 4
Neuroscience. 2021  PMID: 33359660
03. Small Molecule Inhibitors Targeting Gαi2 Protein Attenuate Migration of Cancer Cells
Cancers. 2020  PMID: 32575572
04. Three-dimensional growth of breast cancer cells potentiates the anti-tumor effects of unacylated ghrelin and AZP-531
eLife. 2020  PMID: 32667883
05. Wnts regulate planar cell polarity via heterotrimeric G protein and PI3K signaling
J Cell Biol. 2020  PMID: 32805026
06. The COMMD3/8 complex determines GRK6 specificity for chemoattractant receptors
J Exp Med. 2019  PMID: 31088898
07. VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1
J Cell Sci. 2019 2019  PMID: 31562188
08. Mechanoactivation of the angiotensin II type 1 receptor induces β‐arrestin‐biased signaling through Gαi coupling
J Cell Biochem. 2018  PMID: 29231251
09. Estrogen deficiency compromised the β 2 AR-Gs/Gi coupling: implications for arrhythmia and cardiac injury
Pflugers Arch. 2018  PMID: 29297096
10. Biased antagonism of CXCR4 avoids antagonist tolerance
Sci Signal. 2018   PMID: 30327409
11. Acupuncture Upregulates G Protein Coupled Activity in Samp8 Mice
Acupunct Med. 2017  PMID: 28450287
12. Gαi is required for carvedilol-induced β1 adrenergic receptor β-arrestin biased signaling
Nat Commun. 2017  PMID: 29167435
13. Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor
Thromb Res. 2017  PMID: 28609704
14. Wnt-5a/Frizzled9 Receptor Signaling through the Gαo-Gβγ Complex Regulates Dendritic Spine Formation
J Biol Chem. 2016  PMID: 27402827
15. Targeting brain tumor cAMP: the case for sex-specific therapeutics
Front. Pharmacol. 2015  PMID: 26283963
16. Rapid Actions of Xenoestrogens Disrupt Normal Estrogenic Signaling

Steroids. 2014  PMID: 24269739

17. The lipoprotein receptor LRP1 modulates sphingosine-1-phosphate signaling and is essential for vascular development
Development. 2014  PMID: 25377550
18. Neuronal NF1/RAS regulation of cyclic AMP requires atypical PKC activation
Hum Mol Genet. 2014  PMID: 25070947
19. Fatty Acid-binding Protein 5 (FABP5) Regulates Cognitive Function Both by Decreasing Anandamide Levels and by Activating the Nuclear Receptor Peroxisome Proliferator-activated Receptor ß/d (PPARß/d) in the Brain
J Biol Chem. 2014  PMID: 24644281
20. Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin
Mol Biol Cell. 2014  PMID: 25187647
21. Canonical and noncanonical g-protein signaling helps coordinate actin dynamics to promote macrophage phagocytosis of zymosan
Mol Cell Biol. 2014  PMID: 25225330
22. Identification of novel signalling roles and targets for Galpha and Gbetagamma downstream of the insulin-like growth factor 1 receptor in vascular smooth muscle cells
Biochem J. 2013  PMID: 23186281
23. Heterotrimeric Galpha(i) proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis
Biochim Biophys Acta. 2012  PMID: 21255617
24. CXCR4 activation defines a new subgroup of Sonic hedgehog-driven medulloblastoma
Cancer Res. 2012  PMID: 22052462
25. Regulation of Estradiol and Progesterone Production by CRH-R1 and -R2 Is through Divergent Signaling Pathways in Cultured Human Placental Trophoblasts
Endocrinology. 2012  PMID: 22865371
26. CRH activation of different signaling pathways results in differential calcium signaling in human pregnant myometrium before and during labor
J Clin Endocrinol Metab. 2012  PMID: 22869609
27. Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-à interactions with G protein-ài and caveolin I
Steroids. 2012  PMID: 22230296
28. WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins
Cell Signal. 2011  PMID: 21070854
29. Cannabinoids inhibit insulin receptor signaling in pancreatic β-cells
Diabetes.  2011  PMID: 21346174
30. PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades
Nat Commun. 2011  PMID: 22186894
31. Na/H exchanger regulatory factors control parathyroid hormone receptor signaling by facilitating differential activation of G(alpha) protein subunits
J Biol Chem. 2010  PMID: 20562104
32. Beta-arrestin- but not G protein-mediated signaling by the “decoy” receptor CXCR7
Proc Natl Acad Sci U S A. 2010  PMID: 20018651