Rap1 Pull-Down ActivationAssay Kit
Cat. #81401
Introduction
A.BackgroundSmall GTPases are a super-family of cellular signalingregulators. The small Ras-like GTPase Rap1 is an evolutionaryconserved protein that originally gained interest because of itscapacity to revert the morphological phenotype of Ras-transformedfibroblasts. Rap1 is regulated by a large number of stimuli thatinclude growth factors and cytokines, but also physical force andosmotic stress. Rap1 was shown to regulate multiple basic cellularprocesses. The best studied aspect of Rap1 function in endothelialcells involved its role in regulation of cell-cell junctionformation and remodeling.
Rap1 Activation Assay Kit is based on the configuration-specificmonoclonal antibody that specifically recognizes Rap1-GTP, but notRap1-GDP. Given the high affinity of monoclonal antibodies to theirantigens, the activation assay could be performed in a short time.This assay provides the reliable results with consistentreproducibility.
The anti-Rap1-GTP monoclonal antibody can also be used tomonitor the activation of Rap1 in cells and in tissues byimmunohistochemistry.
B. AssayPrincipleThe Rap1 Activation Assay Kit uses configuration-specificanti-Rap1-GTP Mouse monoclonal antibody to measure Rap1-GTP levelsin cell extracts or in vitro GTPγS loading Rap1 activation assays.Anti-Rap1-GTP mouse monoclonal antibody is first incubated withcell lysates containing Rap1-GTP. Next, the GTP-bound Rap1 ispulled down by protein A/G agarose. Finally, the precipitatedRap1-GTP is detected through immunoblot analysis using Anti-Rap1Rabbit Polyclonal Antibody.
C. KitComponents1. Anti-Rap1-GTP Mouse Monoclonal Antibody (Cat. # 26912): Onevial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol.This antibody specifically recognizes Rap1-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, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5%Triton X-100.
4. Anti-Rap1 Rabbit Polyclonal Antibody (Cat. # 21156): 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 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 Supplied1. 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 ResultsThe following figure demonstrates example results seen with theRap1 Activation Assay Kit. For reference only.
Rap1 ActivationAssay. Purified Rap1 proteins were immunoprecipitatedafter treated with GDP (lane 1) or GTPγS (lane 2).Immunoprecipitation was done with the anti-Rap1-GTP monoclonalantibody (Cat. No. 26912). Immunoblot was with an anti-Rap1polyclonal antibody (Cat. # 21156).
AssayProcedure
A.Reagent Preparation1XAssay/Lysis Buffer: Mix the 5X Stock (Cat. # 30301)briefly and dilute to 1X in deionized water. Just prior to usage,add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, or10 µg/mL aprotinin.
B.Sample PreparationAdherent Cells1. 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 Cells1. 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 NegativecontrolsNote: In vivo stimulation ofcells will activate approximately 10% of the available Rap1,whereas in vitro GTPγS protein loading will activate nearly 90% ofRap1.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Rap1protein) 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 Protein1. 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-Rap1-GTP antibody (Cat. # 26912).
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 Analysis1. 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-Rap1 Rabbit PolyclonalAntibody (Cat. # 21156), which is freshly diluted 1: 50~500(depending on the amount of Rap1 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.
武汉费斯德主营：G蛋白活性抗体，癌症点突变抗体，非乙酰化CAMP和CGMP试剂，抗体定制服务等。