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Rac Pull-Down ActivationAssay Kit

Cat. #80501

Introduction

A.Background

Small GTPases are a super-family of cellular signalingregulators. Rac belongs to the Rho sub-family of GTPases thatregulate cell motility, cell division, and gene transcription. GTPbinding increases the activity of Rac, and the hydrolysis of GTP toGDP renders it inactive.

Currently the activation of Rac proteins is assayed with thebinding of GTP-bound Rac to the p21-binding domain (PBD) ofp21-activated protein kinase (PAK). This method is based on theobservation that the active, GTP-bound Rac could bind to the PBD ofPAK. However, the reproducibility of this method is poor. This ispartially due to the relatively quick hydrolysis of GTP to GDPduring the assay procedure, and the low binding affinity of PBD toRac-GTP.

The Rac Activation Assay Kit is based on theconfiguration-specific monoclonal antibody that specificallyrecognizes Rac-GTP, but not Rac-GDP. Given the high affinity ofmonoclonal antibodies to their antigens, the activation assay couldbe performed in a much shorter time. This assay provides thereliable results with consistent reproducibility.

These anti-Rac-GTP monoclonal antibody can also be used tomonitor the activation of Rac in cells and in tissues byimmunohistochemistry.

B. AssayPrinciple

The Rac Activation Assay Kit uses configuration-specificanti-Rac-GTP Mouse monoclonal antibody to measure Rac-GTP levels incell extracts or in vitro GTPγS loading Rac activation assays.Anti-Rac-GTP mouse monoclonal antibody is first incubated with celllysates containing Rac-GTP. Next, the GTP-bound Rac is pulled downby protein A/G agarose. Finally, the precipitated Rac-GTP isdetected through immunoblot analysis using Anti-Rac RabbitPolyclonal Antibody.

The anti-Rac-GTP monoclonal antibody can also be used to monitorthe activation of Rac in cells and in tissues byimmunohistochemistry.

C. KitComponents

1. Anti-Rac-GTP Mouse Monoclonal Antibody (Cat. # 26903): Onevial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol.This antibody specifically recognizes Rac-GTP from allvertebrates.

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 of250 mM Tris-HCl, pH 8, 750mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5%Triton X-100.

4. Anti-Rac Rabbit Polyclonal Antibody (Cat. # 21003): One vial– 50 µL (1mg/mL) in PBS, pH 7.4, contained 50% glycerol.

5. 100X GTPγS (Cat. # 30303): One vial – 50 µl at 10 mM, use 5µL 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 µLof 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) inPBS, 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, pH7.4, 0.15 M NaCl, 0.05%  Tween-20)

9. Immunoblotting blocking buffer (TBST containing 5% Non-fatDry Milk or 3% BSA)

10. ECL Detection Reagents

E.Example Results

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

RacActivation Assay. MEFcells were treated with (lane 2) or without (lane 1) PDGF. Celllysates were incubated with an anti-Rac-GTP monoclonal antibody(Cat. # 26903) (top panel). The precipitated active Rac wasimmunoblotted with an anti-Rac rabbit polyclonal antibody (Cat. #21003). The bottom panel shows the Western blot with anti-Rac ofthe cell lysates used (5% of that used in the toppanel).

AssayProcedure

A.Reagent Preparation

1X Assay/Lysis Buffer: Mixthe 5X Stock (Cat. # 30301) briefly and dilute to 1X in deionizedwater. Just prior to usage, add protease inhibitors such as 1 mMPMSF, 10 µg/mL leupeptin, or 10 µg/mL aprotinin.

B.Sample PreparationAdherent Cells

1. Culture cells (one 10-cm plate, ~107 cells)to approximately 80-90% confluence. Stimulate the cells withactivator or inhibitor as desired.

2. Aspirate the culture media and wash twice with ice-coldPBS.

3. Completely remove the final PBS wash and add ice-cold 1XAssay/Lysis Buffer (See Reagent Preparation) to the cells (0.5-1 mLper 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 cellscraper.

6. Transfer the lysates to appropriate size tubes and place onice.

7. If nuclear lysis occurs, the cell lysates may become viscousand difficult to pipette. If this occurs, lysates can be passedthrough a 27½-gauge syringe needle 3-4 times to shear the genomicDNA.

8. Clear the lysates by centrifuging at 12,000 x g and 4°C for10 minutes.

9. Collect the supernatant and store the sample (~1-2 mg oftotal protein) on ice for immediate use, or snap freeze and storeat -70°C for future use.

Adherent Cells

1. Culture cells and stimulate with activator or inhibitor asdesired.

2. Perform a cell count and then pellet the cells throughcentrifugation.

3. Aspirate the culture media and wash twice with ice-coldPBS.

4. Completely remove the final PBS wash and add ice-cold 1XAssay/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 themon ice.

9. Collect the supernatant and store sample on ice for immediateuse, or snap freeze and store at -70°C for future use.

C. Invitro GTPγS/GDP Protein for Positive and Negativecontrols

Note: In vivo stimulation ofcells will activate approximately 10% of the available Rac, whereasin vitro GTPγS protein loading will activate nearly 90% ofRac.

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

2. To each tube, add 20 µL of 0.5 M EDTA (final concentration of20 mM).

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

4. Negative control: add 5 µL of 100 X GDP (Cat. # 30304) to the2nd tube.

5. Incubate both tubes at 30°C for 30 minutes withagitation.

6. Stop loading by placing the tubes on ice and adding 32.5 µLof 1 M MgCl2 (finalconcentration of 60 mM).

D.Affinity Precipitation of Activated G Protein

1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of totalcellular protein) to a microcentrifuge tube.

2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (SeeReagent Preparation).

3. Add 1 µL anti-Rac-GTP antibody (Cat. # 26903).

4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) byresuspending through vertexing or titrating.

5. Quickly add 20 µL of resuspended bead slurry to abovetube.

6. Incubate the tube at 4°C for 1 hour with gentleagitation.

7. Pellet the beads through centrifugation at 5,000 x g for 1min.

8. Aspirate and discard the supernatant (making sure not todisturb 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 throughcentrifugation and carefully remove all the supernatant.

11. Resuspend the bead pellet in 20 µL of 2X reducing SDS- PAGEsample 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 polyacrylamidegel (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’sinstructions.

3. Transfer the gel proteins to a PVDF or nitrocellulosemembrane following the manufacturer’s instructions.

Note: Steps 4-11 are at roomtemperature with agitation

4. Following electroblotting, immerse the PVDF membrane in ****Methanol for 15 seconds, and then allow it to dry at roomtemperature for 5 minutes.

Note: If Nitrocellulose isused instead of PVDF, step 4 Should be skipped.

5. Block the membrane with 5% non-fat dry milk or 3% BSA in TBSTfor 1 hr at room temperature with constant agitation.

6. Wash the blotted membrane three times with TBST, 5 minuteseach time.

7. Incubate the membrane with Anti-Rac Rabbit PolyclonalAntibody (Cat. # 21003), which is freshly diluted 1: 50~500(depending on the amount of Rac proteins in your sample) in 5%non-fat dry milk or 3% BSA in TBST, for 1-2 hr at room temperaturewith constant agitation or at 4°C overnight.

8. Wash the blotted membrane three times with TBST, 5 minuteseach time.

9. Incubate the membrane with a secondary antibody (Cat. #29002), which is freshly diluted 1: 1000 in 5% non-fat dry milk or3% BSA in TBST, for 1 hr at room temperature with constantagitation.

10. Wash the blotted membrane three times with TBST, 5 minuteseach time.

11. Use the detection method of your choice such as ECL.