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RheB Pull-Down Activation Assay Kit

RheB Pull-Down Activation Assay Kit
5/5

$737.00

Cat.#:  81201

   Size:   30 Assays

In Stock

          Product Description          

RheB Pull-Down Activation Assay Kit

Cat. # 81201

Introduction

A. Background
Small GTPases are a super-family of cellular signaling regulators. RheB is a member of the Ras-superfamily GTPases. RheB has been shown to interact with C-Raf, Mammalian target of rapamycin (mTOR), TSC2, Ataxia telangiectasia mutated (ATM), KIAA1303 and Ataxia telangiectasia and Rad3 related. Currently there is no direct assay to measure the activation of RheB GTPases.
The RheB Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes RheB-GTP, but not RheB-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a short time. This assay provides the reliable results with consistent reproducibility. These anti-RheB-GTP monoclonal antibodies can also be used to monitor the activation of RheB in cells and in tissues by immunohistochemistry.
B. Assay Principle
The RheB Activation Assay Kit uses configuration-specific anti-RheB-GTP Mouse monoclonal antibody to measure RheB-GTP levels in cell extracts or in vitro GTPγS loading RheB activation assays. Anti-RheB-GTP mouse monoclonal antibody is first incubated with cell lysates containing RheB-GTP. Next, the GTP-bound RheB is pulled down by protein A/G agarose. Finally, the precipitated RheB-GTP is detected through immunoblot analysis using Anti-RheB Rabbit Polyclonal Antibody.
The anti-RheB-GTP monoclonal antibody can also be used to monitor the activation of RheB in cells and in tissues by immunohistochemistry.
C. Kit Components
1. Anti-RheB-GTP Mouse Monoclonal Antibody (Cat. # 26910): 30 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes RheB-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-RheB Rabbit Polyclonal Antibody (Cat. # 21098): 50 µL (1 mg/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 µg/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 RheB Activation Assay Kit. For reference only.

RheB Activation Assay Kit
RheB Activation Assay. Purified RheB proteins were immunoprecipitated after treated with GDP (lane 1) or GTPγS (lane 2). Immunoprecipitation was done with the anti-RheB-GTP monoclonal antibody (Cat. # 26910). Immunoblot was with an anti-RheB rabbit polyclonal antibody (Cat. # 21098).

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 RheB, whereas in vitro GTPγS protein loading will activate nearly 90% of RheB.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified RheB 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-RheB-GTP antibody (Cat. # 26910).
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 hr 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-RheB Rabbit Polyclonal Antibody (Cat. # 21098), which has been freshly diluted 1:50~500 (depending on the amount of RheB proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 hr 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 is freshly diluted 1:1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 hr 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. Mnk1/2 kinases regulate memory and autism-related behaviours via Syngap1
Brain. 2023   PMID: 36315645
02. Mnk1/2 kinases regulate memory and autism-related behaviours via Syngap1
Brain. 2022  PMID: 36315645
03. NADPH Oxidase 2 Derived Reactive Oxygen Species Promote CD8+ T cell Effector Function
bioRxiv. 2021
04. PIR-B Regulates CD4+ IL17a+ T-Cell Survival and Restricts T-Cell-Dependent Intestinal Inflammatory Responses
Cell Mol Gastroenterol Hepatol. 2021  PMID: 34242819
05. Cardiac-specific deletion of FDPS induces cardiac remodeling and dysfunction by enhancing the activity of small GTP-binding proteins
J Pathol. 2021  PMID: 34467534
06. RNA N6-Methyladenosine Methyltransferase METTL3 Facilitates Colorectal Cancer by Activating the m6A-GLUT1-mTORC1 Axis and Is a Therapeutic Target.
Gastroenterology. 2020  PMID: 33217448
07. CBAP modulates Akt-dependent TSC2 phosphorylation to promote Rheb-mTORC1 signaling and growth of T-cell acute lymphoblastic leukemia
Oncogene. 2019  PMID: 30266989
08. Oncogenic KRAS signaling activates mTORC1 through COUP-TFII-mediated lactate production
EMBO Rep. 2019  PMID:  30988000
09. Ubiquitination of Rheb governs growth factor-induced mTORC1 activation
Cell Res. 2018  PMID:  30514904
10. Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death
Brain. 2017  PMID: 28969371
11. Alteration of protein prenylation promotes spermatogonial differentiation and exhausts spermatogonial stem cells in newborn mice
Sci Rep. 2016  PMID: 27374985
12. Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration
Proc Natl Acad Sci U S A. 2016  PMID: 27647922
13. Suppression of the GTPase-activating protein RGS10 increases Rheb-GTP and mTOR signaling in ovarian cancer cells
Cancer Lett. 2015  PMID:  26319900
14. Altered nutrient response of mTORC1 as a result of changes in REDD1 expression: effect of obesity vs. REDD1 deficiency
J Appl Physiol. 2014   PMID: 24876363
15. Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2
Cancer Res. 2014  PMID: 24398473
16. REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling
Sci Signal. 2014  PMID: 25056877
17. SynGAP Regulates Protein Synthesis and Homeostatic Synaptic Plasticity in Developing Cortical Networks
PLoS One. 2013  PMID: 24391850
18. Rheb/mTORC1 Signaling Promotes Kidney Fibroblast Activation and Fibrosis
J Am Soc Nephrol. 2013  PMID: 23661807