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Ras Pull-Down Activation Assay Kit

Ras Pull-Down Pull-Down Assay Kit
5/5

$737.00

Cat.#:  81101

   Size:   30 Assays

In Stock

          Product Description          

Ras Pull-Down Activation Assay Kit

Cat. # 81101

Introduction

A. Background
Small GTPases are a super-family of cellular signaling regulators. Ras belongs to the Ras sub-family of GTPases that regulate cell growth, cell motility, and gene transcription. GTP binding increases the activity of Ras, and the hydrolysis of GTP to GDP renders it inactive.
Currently the activation of Ras proteins is assayed with the binding of GTP-bound Ras to the Ras-binding domain (RBD) of Raf protein kinase. This method is based on the observation that the active, GTP-bound Ras could bind to the RBD of Raf. However, the reproducibility of this method is poor. This is partially due to the relatively quick hydrolysis of GTP to GDP during the assay procedure, and the low binding affinity of RBD to Ras-GTP.
The Ras Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Ras-GTP, but not Ras-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a much shorter time. This assay provides the reliable results with consistent reproducibility.
B. Assay Principle
The Ras Activation Assay Kit uses configuration-specific anti-Ras-GTP Mouse monoclonal antibody to measure Ras-GTP levels in cell extracts or in vitro GTPγS loading Ras activation assays. Anti-Ras-GTP mouse monoclonal antibody is first incubated with cell lysates containing Ras-GTP. Next, the GTP-bound Ras is pulled down by protein A/G agarose. Finally, the precipitated Ras-GTP is detected through immunoblot analysis using Anti-Ras Rabbit Polyclonal Antibody.
The anti-Ras-GTP monoclonal antibody can also be used to monitor the activation of Ras in cells and in tissues by immunohistochemistry.
C. Kit Components
1. Anti-Ras-GTP Mouse Monoclonal Antibody (Cat. # 26909): 30 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Ras-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-Ras Rabbit Polyclonal Antibody (Cat. # 21021): 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 Ras Activation Assay Kit. For reference only.

Ras Activation Assay Kit
Ras Activation Assay. Purified Ras protein was loaded with GDP (lane 1) or GTPγS (lane 2). These proteins were incubated with an anti-Ras-GTP monoclonal antibody (Cat. # 26905) (top panel). The precipitated active Ras was immunoblotted with an anti-Ras rabbit polyclonal antibody (Cat # 21021). The bottom panel shows the Western blot with anti-Ras of the used Ras-GDP and Ras-GTPγS.

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 Ras, whereas in vitro GTPγS protein loading will activate nearly 90% of Ras.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Ras 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-Ras-GTP antibody (Cat. # 26909).
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-Ras Rabbit Polyclonal Antibody (Cat. # 21021), which has been freshly diluted 1:50~500 (depending on the amount of Ras 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. microRNA-324-3p suppresses the aggressive ovarian cancer by targeting WNK2/RAS pathway
Bioengineered. 2022  PMID: 35549643
02. GPR110 promotes progression and metastasis of triple-negative breast cancer
Cell Death Discov. 2022  PMID: 35614051
03. Protection of dopaminergic neurons in hemiparkinsonian monkeys by flavouring ingredient glyceryl tribenzoate
NeuroImmune Pharm Ther. 2022  PMID: 0
04. KRAS Affects Adipogenic Differentiation by Regulating Autophagy and MAPK Activation in 3T3-L1 and C2C12 Cells
Int J Mol Sci. 2021  PMID: 34948427
05. A RAS inhibitor reduces allergic airway remodeling via regulating IL-33-derived type 2 innate lymphoid cells
Exp Lung Res. 2021  PMID: 34739349
06. A novel terpenoid class for prevention and treatment of KRAS-driven cancers: Comprehensive analysis using in situ, in vitro and in vivo model systems
Mol Carcinog. 2020  PMID: 32291806
07. Glycyrrhetinic acid binds to the conserved P-loop region and interferes with the interaction of RAS-effector proteins
Acta Pharm Sin B. 2019  PMID: 30976491
08. The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2
Mol Cell. 2019  PMID: 30639242
09. Glycyrrhetinic Acid Improves Insulin-Response Pathway by Regulating the Balance between the Ras/MAPK and PI3K/Akt Pathways
Nutrients. 2019  PMID: 30871060
10. Characterization of Novel Murine and Human PDAC Cell Models: Identifying the Role of Intestine Specific Homeobox Gene ISX in Hypoxia and Disease Progression
Transl Oncol. 2019  PMID: 31174057
11. ROS/KRAS/AMPK Signaling Contributes to Gemcitabine-Induced Stem-like Cell Properties in Pancreatic Cancer
Mol Ther Oncolytics. 2019  PMID: 31508487
12. KRAS regulation by small non-coding RNAs and SNARE proteins
Nat Commun. 2019  PMID: 31712554
13. Inhibition of Kirsten-Ras reduces fibrosis and protects against renal dysfunction in a mouse model of chronic folic acid nephropathy
Sci Rep. 2019  PMID: 31570767
14. Desmoplakin maintains gap junctions by inhibiting Ras/MAPK and lysosomal degradation of connexin-43
J Cell Biol. 2018  PMID: 29959233
15. GDF15 promotes the proliferation of cervical cancer cells by phosphorylating AKT1 and Erk1/2 through the receptor ErbB2
J Exp Clin Cancer Res. 2018  PMID: 29636108
16. miR-296-5p suppresses EMT of hepatocellular carcinoma via attenuating NRG1/ERBB2/ERBB3 signaling
J Exp Clin Cancer Res. 2018  PMID: 30486894
17. Lectin BS-I inhibits cell migration and invasion via AKT/GSK-3ß/ß-catenin pathway in hepatocellular carcinoma
J Cell Mol Med. 2018  PMID: 28922551
18. Biased signaling by thyroid-stimulating hormone receptor-specific antibodies determines thyrocyte survival in autoimmunity
Sci Signal. 2018  PMID: 29363585
19. Immunohistochemical evalulation of activated Ras and Rac1 as potential downstream effectors of aquaporin-5 in breast cancer in vivo
Biochem Biophys Res Commun. 2017  PMID: 28958942
20. Inhibition of lung cancer in diethylnitrosamine-induced mice by Rhizoma paridis saponins
Mol Carcinog. 2017  PMID: 27991692
21. Developmental stage-dependent regulation of spine formation by calcium-calmodulin-dependent protein kinase IIa and Rap1
Sci Rep. 2017  PMID: 29042611
22. p120RasGAP Protein Mediates Netrin-1 Protein-induced Cortical Axon Outgrowth and Guidance
J Biol Chem. 2016  PMID: 26710849
23. Sos1 Regulates Macrophage Podosome Assembly and Macrophage Invasive Capacity
J Immunol. 2015  PMID: 26447228
24. H-Ras regulation of TRAIL death receptor mediated apoptosis
Oncotarget. 2014  PMID: 25026275
25. Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models
Bone. 2014  PMID: 24947449
26. Depletion of the transcriptional coactivators megakaryoblastic leukaemia 1 and 2 abolishes hepatocellular carcinoma xenograft growth by inducing oncogene-induced senescence
EMBO Mol Med. 2013  PMID: 23853104
27. JNK inhibitors increase osteogenesis in Nf1-deficient cells
Bone. 2011  PMID: 21964323