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Ran Pull-Down Activation Assay Kit
Cat. # 81701
Introduction
A. BackgroundSmall GTPases are a super-family of cellular signalingregulators. Ran (Ras-related nuclear protein) is a member of theRas-superfamily GTPases. Ran is involved in the transport ofproteins across the nuclear envelope, as well as in microtubuleorganization during mitosis. Currently there is no direct assay tomeasure the activation of Ran GTPases.
The Ran Activation Assay Kit is based on theconfiguration-specific monoclonal antibody that specificallyrecognizes Ran-GTP, but not Ran-GDP. Given the high affinity ofmonoclonal antibodies to their antigens, the activation assay couldbe performed in a short time. This assay provides the reliableresults with consistent reproducibility.
B. Assay PrincipleThe Ran Activation Assay Kit uses configuration-specificanti-Ran-GTP Mouse monoclonal antibody to measure Ran-GTP levels incell extracts or in vitro GTPγS loading Ran activation assays.Anti-Ran-GTP mouse monoclonal antibody is first incubated with celllysates containing Ran-GTP. Next, the GTP-bound Ran is pulled downby protein A/G agarose. Finally, the precipitated Ran-GTP isdetected through immunoblot analysis using Anti-Ran RabbitPolyclonal Antibody.
The anti-Ran-GTP monoclonal antibody can also be used to monitorthe activation of Ran in cells and in tissues byimmunohistochemistry.
C. Kit Components1. Anti-Ran-GTP Mouse Monoclonal Antibody (Cat. # 26915): Onevial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol.This antibody specifically recognizes Ran-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-Ran Rabbit Polyclonal Antibody (Cat. # 21097): 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 theRan Activation Assay Kit. For reference only.
Ran Activation Assay.Purified Ranproteins were loaded as a control (lanes 1) or immunoprecipitatedafter treated with GDP (lane 2) or GTPγS (lane 3).Immunoprecipitation was done with the anti-Ran-GTP monoclonalantibody (Cat. # 26915). Immunoblot was with an anti-Ran polyclonalantibody (Cat. # 21097).
Assay Procedure
A. Reagent Preparation1X Assay/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) toapproximately 80-90% confluence. Stimulate the cells with activatoror 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. In vitro GTPγS/GDP Protein for Positive and NegativecontrolsNote: In vivo stimulation of cells will activate approximately10% of the available Ran, whereas in vitro GTPγS protein loadingwill activate nearly 90% of Ran.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Ranprotein) 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 (final concentration 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-Ran-GTP antibody (Cat. # 26915).
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 room temperature 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 is used instead of PVDF, step 4 Shouldbe 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-Ran Rabbit PolyclonalAntibody (Cat. # 21097), which has been freshly diluted 1: 50~500(depending on the amount of Ran 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.