RhoA/Rac1/CDC42 Activation Assay Combo Kit

Catalog No. ABIN2345083

Product Details

Reactivity: Various Species

Application: Activation (Act)

Sample Type: Cell Lysate

Characteristics: RhoA/Rac1/Cdc42 Activation Assay utilizes Rhotekin RBD and PAK PBD Agarose beads to selectively isolate and pull-down the active form of Rho/Rac/Cdc42 from purified samples or endogenous lysates. Subsequently, the precipitated GTP-Rho or Rac or Cdc42 is detected by western blot analysis using an anti-RhoA or Rac1 or Cdc42 antibody (see Figure 3, Figure 5, Figure 7 and Assay Principle). RhoA/Rac1/Cdc42 Activation Assay Kit provides a simple and fast tool to monitor the activation of RhoA, Rac1 or Cdc42. The kit includes easily identifiable Rhotekin RBD and PAK1 PBD Agarose beads (see Figure 1), pink in color, along with RhoA, Rac1 and Cdc42 Immunoblot Positive Controls for quick Rho small GTPase identification. Each kit provides sufficient quantities to perform 10 assays for each small GTPase. Figure 1:Rhotekin RBD or PAK RBD Agarose beads, in color, are easy to visualize, minimizing potential loss during washes and aspirations.

Components:

1. Rhotekin RBD Agarose : One vial - 400 μL of 50% slurry, 200 μg Rhotekin RBD in PBS containing 50% glycerol. Note: Agarose bead appears pink in color for easy identification, washing, and aspiration.
2. PAK1 PBD Agarose : One vial - 800 μL of 50% slurry, 400 μg PAK1 PBD in PBS containing 50% glycerol. Note: Agarose bead appears pink in color for easy identification, washing, and aspiration.
3. 100X GTPγS : Two vials - 2 x 50 μL of 10 mM GTPγS dissolved in sterile water.
4. 100X GDP : Two vials - 2 x 50 μL of 100 mM GDP dissolved in sterile water.
5. 5X Assay/Lysis Buffer : Two bottles - 2 x 30 mL of 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% NP-40, 50 mM MgCl2, 5 mM EDTA, 10% Glycerol.
6. Anti-RhoA, Mouse Monoclonal : One vial - 40 μL in PBS, pH 7.4, 0.05% NaN3, 0.1% BSA.
7. Anti-Rac1, Mouse Monoclonal : One vial - 40 μL in PBS, pH 7.4, 0.05% NaN3, 0.1% BSA. Note: This monoclonal reacts with human, mouse, and rat Rac1, it also has slight cross- reactivity with Rac2. Additional unknown higher MW proteins may be detected in some preparations.
8. Anti-Cdc42, Mouse Monoclonal : One vial - 40 μL in PBS, pH 7.4, 0.05% NaN3, 0.1% BSA.
9. RhoA Immunoblot Positive Control : One vial - 100 μL of partially purified, recombinant RhoA from E. coli (provided ready-to-use in 1X reducing SDS-PAGE Sample Buffer, pre-boiled).
10. Rac1 Immunoblot Positive Control : One vial - 100 μL of partially purified, recombinant Rac1 from E. coli (provided ready-to-use in 1X reducing SDS-PAGE Sample Buffer, pre-boiled).
11. Cdc42 Immunoblot Positive Control : One vial - 100 μL of partially purified, recombinant Cdc42 from E. coli (provided ready-to-use in 1X reducing SDS-PAGE Sample Buffer, pre-boiled). 4

Material not included:

1. Stimulated and non-stimulated cell lysates
2. RhoA, Rac1 and/or Cdc42 activators
3. Protease inhibitors
4. 0.5 M EDTA in water
5. 1 M MgCl2
6. 30 °C incubator or water bath
7. 4 °C tube rocker or shaker
8. 2X reducing SDS-PAGE sample buffer
9. Electrophoresis and immunoblotting systems
10. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05 % Tween-20)
11. Immunoblotting blocking buffer (TBST containing 5 % Non-fat Dry Milk)
12. PVDF or nitrocellulose membrane
13. Secondary Antibody
14. ECL Detection Reagents

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Safe non-radioactive assay format
• Colored agarose beads allow visual check
• Fast results: 1 hour plus electrophoresis/blotting time

Assay Time: 1 h

Reagent Preparation:

1X Assay/Lysis Buffer: Mix the 5X Stock briefly and dilute to 1X in deionized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin.

Sample Preparation:

Note: It is advisable to use fresh cell lysates because GTP-small GTPase is quickly hydrolyzed to GDP-small GTPase, frozen lysates stored at -70 °C may be used. Performing steps at 4 °C or on ice may reduce hydrolysis. Avoid multiple freeze/thaw cycles of lysates.

I. Adherent Cells

1. Culture cells to approximately 80-90 % confluence. Stimulate cells with RhoA, Rac1 or Cdc42 activator(s) 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 to the cells (0.5 - 1 mL per 100 mm 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. 5
7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this occurs, lysates can be passed through a 271/2-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifugation for 10 minutes (14,000 x g at 4 °C).
9. Collect the supernatant and store samples on ice for immediate use, or snap freeze and store at - 70 °C for future use.
10. Proceed to GTPγS/GDP Loading for positive and negative controls, or Pull-Down Assay.

II. Suspension Cells

1. Culture cells and stimulate with RhoA, Rac1 or Cdc42 activator(s) as desired.
2. Perform a cell count, and then pellet the cells by 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 to the cell pellet (0.5 - 7 1 mL per 1 x 10 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place on ice.
7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this occurs, lysates can be passed through a 271/2-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifugation for 10 minutes (14,000 x g at 4 °C).
9. Collect the supernatant and store samples on ice for immediate use, or snap freeze and store at - 70 °C for future use.
10. Proceed to GTPγS/GDP Loading for positive and negative controls, or Pull-Down Assay.

Assay Procedure:

Important Note: Before running any Small GTPase pulldown assay, it is always a good practice to run a Western Blot directly on the cell lysate using the antibody provided in this kit. For example: load 5 μg, 10 μg and 20 μg of lysate onto an SDS-PAGE gel, transfer and blot. When proceeding with the pulldown assay, use 100-times the amount of lysate that gave you a clear band of your desired small GTPase in the direct Western blot. For example: if the 5 μg band was faint but the 10 μg band was clear and strong, use 100 x 10 μg = 1 mg of lysate in the assay. Using sufficient lysate in the pulldown assay is critical to success.

I. GTPγS/GDP Loading (Positive and Negative Controls) Note: Samples that will not be GTPγS/GDP loaded may be kept on ice during the loading of controls.

1. Aliquot 0.5 - 1 mL of each cell lysate to two microcentrifuge tubes. Note: Typical protein content/sample is > 0.5 mg.
2. Adjust the volume of each sample to 1 mL with 1X Assay Lysis Buffer. 6
3. Add 20 μL of 0.5 M EDTA to each sample.
4. Add 10 μL of 100X GTPγS to one tube (positive control) and 10 μL of 100X GDP to the other tube (negative control). Mix and label each tube appropriately.
5. Incubate the tubes for 30 minutes at 30 °C with agitation.
6. Stop the loading by adding 65 μL of 1 M MgCl2 to each tube. Mix and place tubes on ice.
7. Continue with Pull-Down assay.

II. Small GTPase Pull-Down Assay

1. Aliquot 0.5 - 1 mL of cell lysate (treated with small GTPase activators or untreated) to a microcentrifuge tube.
2. Adjust the volume of each sample to 1 mL with 1X Assay Lysis Buffer.
3. Thoroughly resuspend the Rhotekin RBD or PAK PBD Agarose bead slurry by vortexing or titurating.
4. Quickly add 40 μL of resuspended bead slurry to each tube (including GTPγS/GDP controls).
5. Incubate the tubes at 4 °C for 1 hour with gentle agitation.
6. Pellet the beads by centrifugation for 10 seconds at 14,000 x g.
7. Aspirate and discard the supernatant, making sure not to disturb/remove the bead pellet.
8. Wash the bead 3 times with 0.5 mL of 1X Assay Buffer, centrifuging and aspirating each time.
9. After the last wash, pellet the beads and carefully remove all the supernatant.
10. Resuspend the bead pellet in 40 μL of 2X reducing SDS-PAGE sample buffer.
11. Boil each sample for 5 minutes.
12. Centrifuge each sample for 10 seconds at 14,000 x g.

III. Electrophoresis and Transfer

1. Load 20 μL/well of pull-down supernatant to a polyacrylamide gel. Also, it's recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3). Note: If desired, 10 μL/well of RhoA, Rac1 or Cdc42 Immunoblot Positive Control (provided ready-to-use, pre-boiled) can be added as an immunoblot positive control.
2. Perform SDS-PAGE as per the manufacturer's instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane as per the manufacturer's instructions.

IV. Immunoblotting and Detection (all steps are at room temperature, with agitation)

1. Following the electroblotting step, 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, this step should be skipped. 7
2. Block the membrane with 5 % non-fat dry milk in TBST for 1 hr at room temperature with constant agitation. Incubate the membrane with Anti-RhoA, Rac1 or Cdc42 Antibody, freshly diluted 1:200 to 1:1000 in 5 % non-fat dry milk/TBST, for 1-2 hr at room temperature with constant agitation. Note: To conserve antibody, incubations should be performed in a plastic bag.
3. Wash the blotted membrane three times with TBST, 5 minutes each time.
4. Incubate the membrane with a secondary antibody (e.g. Goat Anti-Mouse IgG, HRP- conjugate), freshly diluted in 5 % non-fat dry milk/TBST, for 1 hr at room temperature with constant agitation.
5. Wash the blotted membrane three times with TBST, 5 minutes each time.
6. Use the detection method of your choice. We recommend enhanced chemiluminescence reagents from Pierce.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: -20 °C

Storage Comment: Store all kit components at -20°C. The 5X Assay/Lysis Buffer may be stored at either -20°C or 4°C. Avoid multiple freeze/thaw cycles.

References

Giralt, Coura, Girault: "Pyk2 is essential for astrocytes mobility following brain lesion." in: Glia, Vol. 64, Issue 4, pp. 620-34, (2016) (PubMed).

Le, Cazares, Mouw, Chatterjee, Macias, Moran, Ramos, Keely, Weaver, Hinck: "Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness." in: The Journal of cell biology, Vol. 212, Issue 6, pp. 707-19, (2016) (PubMed).

Shen, Wang, He, Huang, He, Zhou, Yan, Shen, Shao, Shen, Weng, Lin, Chen: "NMDA receptors participate in the progression of diabetic kidney disease by decreasing Cdc42-GTP activation in podocytes." in: The Journal of pathology, Vol. 240, Issue 2, pp. 149-60, (2016) (PubMed).

Prudent, Popgeorgiev, Gadet, Deygas, Rimokh, Gillet: "Mitochondrial Ca(2+) uptake controls actin cytoskeleton dynamics during cell migration." in: Scientific reports, Vol. 6, pp. 36570, (2016) (PubMed).

Barbati, Alessandri, Vomero, Vona, Colasanti, Vacirca, Camerini, Crescenzi, Pendolino, Truglia, Conti, Garofalo, Sorice, Pierdominici, Valesini, Malorni, Ortona: "Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes." in: Journal of autoimmunity, Vol. 58, pp. 78-89, (2015) (PubMed).

Toyoda, Fukuda, Sanui, Tanaka, Yamamichi, Atomura, Maeda, Tomokiyo, Taketomi, Uchiumi, Nishimura: "Grp78 Is Critical for Amelogenin-Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line." in: Journal of cellular physiology, Vol. 231, Issue 2, pp. 414-27, (2015) (PubMed).

Mori, Izawa, Tanaka: "Smad3 deficiency leads to mandibular condyle degradation via the sphingosine 1-phosphate (S1P)/S1P3 signaling axis." in: The American journal of pathology, Vol. 185, Issue 10, pp. 2742-56, (2015) (PubMed).

Daniels, Holman, Cruz-Orengo, Jujjavarapu, Durrant, Klein: "Viral pathogen-associated molecular patterns regulate blood-brain barrier integrity via competing innate cytokine signals." in: mBio, Vol. 5, Issue 5, pp. e01476-14, (2014) (PubMed).

Luis-Ravelo, Antón, Zandueta, Valencia, Pajares, Agorreta, Montuenga, Vicent, Wistuba, De Las Rivas, Lecanda: "RHOB influences lung adenocarcinoma metastasis and resistance in a host-sensitive manner." in: Molecular oncology, Vol. 8, Issue 2, pp. 196-206, (2014) (PubMed).

Khalil, Hanna, Saykali, El-Sitt, Nasrallah, Marston, El-Sabban, Hahn, Symons, El-Sibai: "The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility." in: Experimental cell research, Vol. 321, Issue 2, pp. 109-22, (2014) (PubMed).

Hanna, Khalil, Nasrallah, Saykali, Sobh, Nasser, El-Sibai: "StarD13 is a tumor suppressor in breast cancer that regulates cell motility and invasion." in: International journal of oncology, Vol. 44, Issue 5, pp. 1499-511, (2014) (PubMed).

Quint, Ruan, Pederson, Kassem, Westendorf, Khosla, Oursler: "Sphingosine 1-phosphate (S1P) receptors 1 and 2 coordinately induce mesenchymal cell migration through S1P activation of complementary kinase pathways." in: The Journal of biological chemistry, Vol. 288, Issue 8, pp. 5398-406, (2013) (PubMed).

Nasrallah, Saykali, Al Dimassi, Khoury, Hanna, El-Sibai: "Effect of StarD13 on colorectal cancer proliferation, motility and invasion." in: Oncology reports, Vol. 31, Issue 1, pp. 505-15, (2013) (PubMed).

Wang, Sun, Yang, Luo, Gao, Liu, Qiu, Wang: "DEK depletion negatively regulates Rho/ROCK/MLC pathway in non-small cell lung cancer." in: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, Vol. 61, Issue 7, pp. 510-21, (2013) (PubMed).

Yu, Zhang, Liu, Li, Yu, Wu, Huang: "RhoGDI SUMOylation at Lys-138 increases its binding activity to Rho GTPase and its inhibiting cancer cell motility." in: The Journal of biological chemistry, Vol. 287, Issue 17, pp. 13752-60, (2012) (PubMed).

Baranwal, Wang, Rathinam, Lee, Jin, McGoey, Pylayeva, Giancotti, Blobe, Alahari: "Molecular characterization of the tumor-suppressive function of nischarin in breast cancer." in: Journal of the National Cancer Institute, Vol. 103, Issue 20, pp. 1513-28, (2011) (PubMed).

Ma, Jham, Hu, Friedman, Basile, Molinolo, Sodhi, Montaner: "Viral G protein-coupled receptor up-regulates Angiopoietin-like 4 promoting angiogenesis and vascular permeability in Kaposi's sarcoma." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, Issue 32, pp. 14363-8, (2010) (PubMed).

Xu, Yuan, Mak, Pardanaud, Caunt, Kasman, Larrivée, Del Toro, Suchting, Medvinsky, Silva, Yang, Thomas, Koch, Alitalo, Eichmann, Bagri: "Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3." in: The Journal of cell biology, Vol. 188, Issue 1, pp. 115-30, (2010) (PubMed).

Tian, Jacobo, Billing, Rozkalne, Gage, Anagnostou, Pavenstädt, Pavenstaedt, Hsu, Schlondorff, Ramos, Greka: "Antagonistic regulation of actin dynamics and cell motility by TRPC5 and TRPC6 channels." in: Science signaling, Vol. 3, Issue 145, pp. ra77, (2010) (PubMed).

Liu, Su, Wiznitzer, Epifano, Ladisch: "Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts." in: Glycobiology, Vol. 18, Issue 8, pp. 593-601, (2008) (PubMed).

Target Details

Background: Small GTP-binding proteins (or GTPases) are a family of proteins that serve as molecular regulators in signaling transduction pathways. RhoA, Rac1 and Cdc42, 21 kDa proteins, belong to the family of Rho GTPases regulates a variety of biological response pathways that include cell motility, cell division, gene transcription, and cell transformation. Like other small GTPases, RhoA, Rac1 and Cdc42 regulate molecular events by cycling between an inactive GDP-bound form and an active GTP- bound form. In its active (GTP-bound) state, RhoA binds specifically to the Rho-binding domain (RBD) of Rhotekin, and Rac1 or Cdc42 binds specifically to the p21-binding domain (PBD) of p21- activated protein kinase (PAK) to control downstream signaling cascades.