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Rab5B Pull-Down Activation Assay Kit

Rab5B Pull-Down Activation Assay Kit
5/5

$737.00

Cat.#:  83401

   Size:   30 Assays

In Stock

          Product Description          

Rab5B Pull-Down Activation Assay Kit

Cat. # 83401

Introduction

A. Background
Small GTPases are a super-family of cellular signaling regulators. Rab5 is a member of the Rab-family GTPases. Rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.
Currently there is no direct assay to measure the activation of Rab5B GTPases.
Rab5B Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rab5-GTP, but not Rab5-GDP, and a Rab5B specific rabbit polyclonal antibody. 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.
B. Assay Principle
The Rab5B Activation Assay Kit uses configuration-specific anti-Rab5-GTP Mouse monoclonal antibody to measure Rab5-GTP levels in cell extracts or in vitro GTPγS loading Rab5 activation assays. Anti-Rab5-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rab5-GTP. Next, the GTP-bound Rab5 is pulled down by protein A/G agarose. Finally, the precipitated Rab5-GTP is detected through immunoblot analysis using Anti-Rab5B Rabbit Polyclonal Antibody.
C. Kit Components
1. Anti-Rab5-GTP Mouse Monoclonal Antibody (Cat. # 26911): 30 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rab5-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-Rab5B Rabbit Polyclonal Antibody (Cat. # 21037): 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 Rab5 Activation Assay Kit. For reference only.
Rab5B Activation Assay Kit Rab5B Activation Assay.Purified His-tagged Rab5B proteins (Cat. #10175) were immunoprecipitated with the anti-Rab5-GTP monoclonal antibody (Cat. #26911) after treated with GDP (lane 1) or GTPγS (lane 2), and was blotted with anti-Rab5B polyclonal antibody (Cat. #21037). Input control is shown in 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.
Adherent 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 Rab5, whereas in vitro GTPγS protein loading will activate nearly 90% of Rab5.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Rab5 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-Rab5-GTP antibody (Cat. # 26911).
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-Rab5B Rabbit Polyclonal Antibody (Cat. # 21037), which is freshly diluted 1:50~500 (depending on the amount of Rab5 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 Downmodulates Alpha-Synuclein-Specific T Cell Responses by Mediating Antigen Trafficking in Dendritic Cells
Front Immunol. 2022  PMID: 35309340
02. Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration
Nat Commun. 2022  PMID: 36130946
03. Assessing rab5 activation in health and disease
Methods Mol Biol.. 2021  PMID: 34453725
04. Role of Endocytosis Proteins in Gefitinib-Mediated EGFR Internalisation in Glioma Cells
Cells. 2021  PMID: 34831480
05. cGMP favors the interaction between APP and BACE1 by inhibiting Rab5 GTPase activity
Sci Rep.. 2020  PMID: 31992816
06. Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth
Signal Transduct Target Ther. 2020  PMID: 33033232
07. Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer’s Disease
Cell Rep.. 2020  PMID: 33238112
08. High glucose-mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities
Br J Pharmacol. 2020  PMID: 32436237
09. NuMA1 promotes axon initial segment assembly through inhibition of endocytosis
J Cell Biol. 2020  PMID: 31727776
10. Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFß receptor endocytosis
FASEB J. 2020  PMID: 32304339
11. ROR1 sustains multivesicular endosomes by interacting with HRS and STAM1
Research Square. 2020
12. TUFT1 promotes metastasis and chemoresistance in triple negative breast cancer through the TUFT1/Rab5/Rac1 pathway
Cancer Cell Int. 2019  PMID: 31572059
13. ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-?B via phosphoinosotide remodeling of endosomes
Nat Commun. 2019  PMID: 31113953
14. GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome
J Am Soc Nephrol. 2018  PMID: 29959197
15. Ulk1 Governs Nerve Growth Factor/TrkA Signaling by Mediating Rab5 GTPase Activation in Porcine Hemagglutinating Encephalomyelitis Virus-Induced Neurodegenerative Disorders
J Virol. 2018  PMID: 29875237
16. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling
Mol Biol Cell. 2016  PMID: 26582392
17. Rin1 restores host phagocytic activity during invasion by Pseudomonas aeruginosa
J Med Microbiol. 2016  PMID: 26902911
18. AKT-independent Reelin signaling requires interactions of heterotrimeric Go and Src
Biochem Biophys Res Commun. 2015   PMID: 26441085
19. Central role for PICALM in amyloid–ß blood–brain barrier transcytosis and clearance
Nat Neurosci. 2015  PMID: 26005850
20. Intracellular Signaling and Trafficking in Cancer: Role of Rab5-GTPase in Migration and Invasion of Breast Cells
FIU. 2015
21. Rab5-mediated VE-cadherin internalization regulates the barrier function of the lung microvascular endothelium
Cell Mol Life Sci. 2015  PMID: 26112597
22. Rab5 Activity Regulates GLUT4 Sorting Into Insulin-Responsive and Non-Insulin-Responsive Endosomal Compartments: A Potential Mechanism for Development of Insulin Resistance
Endocrinology. 2014  PMID: 24932807