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Rab7 Pull-Down Activation Assay Kit

Rab7 Pull-Down Activation Assay Kit
5/5

$737.00

Cat.#:  82501

   Size:   30 Assays

In Stock

          Product Description          

Rab7 Pull-Down Activation Assay Kit

Cat. # 82501

Introduction

A. Background
Small GTPases are a super-family of cellular signaling regulators. The small Ras-like GTPase Rab7 is an evolutionary conserved protein that originally gained interest because of its capacity to revert the morphological phenotype of Ras-transformed fibroblasts. Rab7 is regulated by a large number of stimuli that include growth factors and cytokines, but also physical force and osmotic stress. Rab7 was shown to regulate multiple basic cellular processes. The best studied aspect of Rab7 function in endothelial cells involved its role in regulation of cell-cell junction formation and remodeling.
Rab7 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rab7-GTP, but not Rab7-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.
The anti-Rab7-GTP monoclonal antibody can also be used to monitor the activation of Rab7 in cells and in tissues by immunohistochemistry.
B. Assay Principle
The Rab7 Activation Assay Kit uses configuration-specific anti-Rab7-GTP Mouse monoclonal antibody to measure Rab7-GTP levels in cell extracts or in vitro GTPγS loading Rab7 activation assays. Anti-Rab7-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rab7-GTP. Next, the GTP-bound Rab7 is pulled down by protein A/G agarose. Finally, the precipitated Rab7-GTP is detected through immunoblot analysis using Anti-Rab7 Rabbit Polyclonal Antibody.
C. Kit Components
1. Anti-Rab7-GTP Mouse Monoclonal Antibody (Cat. # 26923): 30 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rab7-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-Rab7 Rabbit Polyclonal Antibody (Cat. # 21069): 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 Rab7 Activation Assay Kit. For reference only.

Rab7 Activation Assay Kit
Rab7 Activation Assay. Purified GST-tagged Rab7 proteins (Cat. #10134) were immunoprecipitated with the anti-Rab7-GTP monoclonal antibody (Cat. No. 26923) after treated with GDP (lane 1) or GTPγS (lane 2), and was blotted with anti-Rab7 polyclonal antibody (Cat. # 21069). Input control is shown in the bottom panel.

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 Rab7, whereas in vitro GTPγS protein loading will activate nearly 90% of Rab7.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Rab7 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-Rab7-GTP antibody (Cat. # 26923).
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-Rab7 Rabbit Polyclonal Antibody (Cat. # 21069), which is freshly diluted 1:50~500 (depending on the amount of Rab7 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. Celastrol directly binds with VAMP7 and RAB7 to inhibit autophagy and induce apoptosis in preadipocytes
Front Pharmacol. 2023   PMID:  36959859
02. Loss of small GTPase Rab7 activation in prion infection negatively affects a feedback loop regulating neuronal cholesterol metabolism
JBC. 2022  PMID: 
03. Celastrol Downmodulates Alpha-Synuclein-Specific T Cell Responses by Mediating Antigen Trafficking in Dendritic Cells
Front Immunol. 2022  PMID: 35309340
04. CD137-CD137L Aggravates Calcification of Vascular Smooth Muscle Cell and Vasculature of ApoE -/- Mice Via Rab7-Mediated Autophagy
J Cardiovasc Transl Res. 2022  PMID: 35763154
05. Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy
Nat Microbiol. 2021  PMID: 34556855
06. FCHSD2 controls oncogenic ERK1/2 signaling outcome by regulating endocytic trafficking
PLoS Biol. 2020  PMID: 32678845
07. Mechanisms underlying endolysosomal deficits mediated by the Parkinson’s disease-related kinase LRRK2
J Parkinsons Dis. 2020  PMID: 33044192
08. Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth
Signal Transduct Target Ther. 2020  PMID: 33033232
09. Interferon-ß-induced miR-1 alleviates toxic protein accumulation by controlling autophagy
eLife. 2019  PMID: 31799933
10. The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A
J Biol Chem. 2019  PMID: 30709905
11. A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium
J Cell Sci., 2019  PMID: 30510112
12. Inhibition of TBC1D5 activates Rab7a and can enhance the function of the retromer cargo-selective complex
J Cell Sci. 2018  PMID: 29777037
13. Temozolomide-Perillyl alcohol conjugate impairs Mitophagy flux by inducing lysosomal dysfunction in non-small cell lung Cancer cells and sensitizes them to irradiation
J Exp Clin Cancer Res. 2018  PMID: 30326943
14. CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection
Autophagy. 2018  PMID: 30165781
15. Rab7-a novel redox target that modulates inflammatory pain processing
Pain. 2017   PMID: 28394828