RhoA 活性检测试剂盒武汉费斯德生物科技有限公司

Small GTPases are a super-family of cellular signaling regulators. RhoA belongs to the Rho sub-family of GTPases that regulate cell motility, cell division, and gene transcription. GTP binding increases the activity of RhoA, and the hydrolysis of GTP to GDP renders it inactive.

Currently the activation of RhoA proteins is assayed with the binding of GTP-bound RhoA to the p21-binding domain (PBD) of p21-activated protein kinase (PAK). This method is based on the observation that the active, GTP-bound RhoA could bind to the PBD of PAK. 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 PBD to RhoA-GTP.

The RhoA Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes RhoA-GTP, but not RhoA-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.

The anti-RhoA-GTP monoclonal antibody can also be used to monitor the activation of RhoA in cells and in tissues by immunohistochemistry.

The RhoA Activation Assay Kit uses configuration-specific anti-RhoA-GTP Mouse monoclonal antibody to measure RhoA-GTP levels in cell extracts or in vitro GTPγS loading RhoA activation assays. Anti-RhoA-GTP mouse monoclonal antibody is first incubated with cell lysates containing RhoA-GTP. Next, the GTP-bound RhoA is pulled down by protein A/G agarose. Finally, the precipitated RhoA-GTP is detected through immunoblot analysis using Anti-RhoA Rabbit Polyclonal Antibody.

1. Anti-RhoA-GTP Mouse Monoclonal Antibody (Cat. # 26904): One vial – 35 μL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes RhoA-GTP from all vertebrates.

2. Protein A/G Agarose (Cat. # 30301): One vial – 600 μL of 50% slurry.

3. 5X Assay/Lysis Buffer (Cat. # 30302): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.

4. Anti-RhoA Rabbit Polyclonal Antibody (Cat. # 21017): One vial – 50 μL (1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.

5. 100X GTPγS (Cat. # 30303): One vial – 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): One vial – 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 mg/mL) in PBS, pH 7.4, contained 50% glycerol.

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

The following figure demonstrates example results seen with the RhoA Activation Assay Kit. For reference only.

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.

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.

1. Aliquot 0.5 mL of cell extract (or 1 μg of purified RhoA protein) into two microcentrifuge tubes.

2. To each tube, add 20 μL of 0.5 M EDTA (final concentration of 20 mM).

3. Positive control: add 5 μL of 100 X GTPγS (Cat. # 30302) to the 1st tube

4. Negative control: add 5 μL of 100 X GDP (Cat. # 30304) to the 2nd tube.

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).

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-RhoA-GTP antibody (Cat. # 26904).

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.

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.

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.

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-RhoA Rabbit Polyclonal Antibody (Cat. # 21017), which is freshly diluted 1: 50~500 (depending on the amount of RhoA 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.

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