污水處理設備 污泥處理設備 水處理過濾器 軟化水設備/除鹽設備 純凈水設備 消毒設備|加藥設備 供水/儲水/集水/排水/輔助 水處理膜 過濾器濾芯 水處理濾料 水處理劑 水處理填料 其它水處理設備
武漢紐斯特生物科技有限公司
武漢紐斯特生物科技有限公司
Configuration-specificMonoclonalAntibodyBasedRab4AActivationAssayKitCatalogNumber：8270120assaysProductDescriptionRab-4AbelongstothesmallGTPaseRabfamily
Configuration-specific Monoclonal Antibody Based
Rab4A Activation Assay Kit
Catalog Number：82701
20 assays
Product Description
Rab-4A belongs to the small GTPase Rab family. It interacts with RAB11FIP1, RABEP1, ZFYVE20,
RUFY1, SGSM1, SGSM2, SGSM3 and other proteins. It participates in protein transport and in
vesicular traffic.
Currently there is no direct assay to measure the activation of Rab4A GTPases.
NewEast Biosciences Rab4A Activation Assay Kit is based on the configuration-specific monoclonal
antibody that specifically recognizes Rab4A-GTP, but not Rab4A-GDP. Given the high affinity of
monoclonal antibodies to their antigens, the activation assay could be performed in a short time. This
assay provides the reliable results with consistent reproducibility.
These anti-Rab4A-GTP monoclonal antibodies can also be used to monitor the activation of Rab4A in
cells and in tissues by immunohistochemistry.
NewEast Biosciences Rab4A Activation Assay Kit provides a simple and fast method to monitor the
activation of Rab4A. Each kit provides sufficient quantities to perform 20 assays.
Assay Principle
NewEast Biosciences Rab4A Activation Assay Kit bases on the configuration-specific anti-Rab4A-GTP
monoclonal antibody to measure the active Rab4A-GTP levels, either from cell extracts or from in vitro
GTPγS loading Rab4A activation assays. Briefly, anti-active Rab4A mouse monoclonal antibody will
be incubated with cell lysates containing Rab4A-GTP. The bound active Rab4A will then be pulled
down by protein A/G agarose beads. The precipitated active Rab4A will be detected by immunoblot
analysis using anti-Rab4A rabbit polyclonal antibody.
Kit Components
1. Anti-active Rab4A, Mouse Monoclonal Antibody (Catalog No. 26921): One vial – 22 µL (1
mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody
specifically recognizes Rab4A-GTP from all vertebrates.
2. Protein A/G Agarose (Catalog No. 30301): One vial – 400 µL of 50% slurry.
3. 5X Assay/Lysis Buffer (Catalog No. 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-Rab4A, Rabbit Polyclonal Antibody (Catalog No. 21158): One vial – 100 µL (1 mg/ml)
in PBS, pH 7.4, contained 50% glycerol.
5. 100 X GTPγS (Catalog No. 30303): One vial –100 μl at 10 mM, use 5 µL of GTPγS for
GTP-labeling of 0.5 mL of cell lysate.
6. 100 X GDP (Catalog No. 30304): One vial –100 μl at 100 mM, use 5 µL of GDP for
GDP-labeling of 0.5 mL of cell lysate.
Storage
Store all kit components at 4oC until their expiration dates.
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, pH8.0
5. 1 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. PVDF or nitrocellulose membrane
11. Secondary Antibody
12. ECL Detection Reagents
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
Adherent Cells
1. Culture cells (one 10-cm plate, ~ 107cells) to approximately 80-90% confluence. Stimulate 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 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 very viscous and difficult to pipette. If this
occurs, lysates can be passed through a 27?-gauge syringe needle 3-4 times to shear the
genomic DNA.
8. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).
9. Collect the supernatant and store samples (~1-2 mg of total proteins) on ice for immediate use,
or snap freeze and store at - 70 °C for future use.
Suspension Cells
1. Culture cells and stimulate with activator or inhibitor 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 – 1 mL per 1 x 107cells).
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 27?-gauge syringe needle 3-4 times to shear the
genomic DNA.
8. Clear the lysates by centrifugation for 10 minutes (12,000 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.
In vitro GTPγS/GDP Protein Loading for positive and negative controls
Note: In vivo stimulation of cells will activate approximately 10% of the available Rab4A, whereas
in vitro GTPγS protein loading will activate nearly 90% of the Rab4A.
Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 μg of purified Rab4A protein).
To each tube, add 20 µl of 0.5 M EDTA (to 20 mM final concentration).
Add 5 µl of 100 X GTPγS (to 100 µM, final concentration) to one tube (positive control).
Add 5 µl of 100 X GDP (to 1 mM, final concentration) to the second tube (negative control).
Incubate the tubes at 30°C for 30 minutes with agitation.
Stop loading by placing the tubes on ice and adding 32.5 µl of 1 M MgCl2 (to 60 mM, final
concentration).
Assay Procedure
I. Active Rab4A Pull-Down Assay
1. Aliquot 0.5 – 1 mL of cell lysate to a microcentrifuge tube.
2. Adjust the volume of each sample to 1 mL with 1X Assay/Lysis Buffer.
3. Add 1 μl anti-active Rab4A monoclonal antibody to the tube.
4. Thoroughly resuspend the protein A/G Agarose bead slurry by vortexing or titurating.
5. Quickly add 20 µL of resuspended bead slurry to each tube.
6. Incubate the tubes at 4 °C for 1 hour with gentle agitation.
7. Pellet the beads by centrifugation for 1 min at 5,000 x g.
8. Aspirate and discard the supernatant, making sure not to disturb/remove the bead pellet.
9. Wash the bead 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each
time.
10. After the last wash, pellet the beads and carefully remove all the supernatant.
11. Resuspend the bead pellet in 20 µL of 2X reducing SDS-PAGE sample buffer.
12. Boil each sample for 5 minutes.
13. Centrifuge each sample for 10 seconds at 5,000 x g.
II. Electrophoresis and Transfer
1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (17%). Also, it’s
recommended to include a pre-stained MW standard (as an indicator of a successful transfer
instep 3).
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.
III. Immunoblotting and Detection (all steps are at room temperature, with agitation)
1. Following the electroblotting step, immerse the PVDF membrane in 99% 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.
2. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature
with constant agitation.
Incubate the membrane with anti-Rab4A polyclonal antibody, freshly diluted 1:50~1000
(depending on the amount of Rab4A proteins in your samples) in 5% non-fat dry milk or 3%
BSA/TBST, for 1-2 hr at room temperature with constant agitation or at 4oC overnight.
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-Rabbit IgG, HRP-conjugate),
freshly diluted 1:1000 in 5% non-fat dry milk or 3% BSA/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 such as ECL.
Example of Results
The following figure demonstrates typical results seen with NewEast Biosciences Rab4A Activation
Assay Kit. One should use the data below for reference only.
Rab4A activation assay. Purified GST-tagged Rab4A proteins (Cat. #10130) were immunoprecipitated
with the anti-active Rab4A monoclonal antibody (Cat. No. 26921) after treated with GDP (lane 1) or
GTPγS (lane 2), and was blotted with anti- Rab4A polyclonal antibody(Cat. No. 21067). Input control is
shown in the bottom panel.
| Related Products | |||
| Catalog# | Name | Size | Price |
| Active Rab4A-GTP Monoclonal Antibody | 30 μL | ￥4800 | |
| Rab4A Activation Assay Kit | 20 assays | ￥6800 | |
您感興趣的產品PRODUCTS YOU ARE INTERESTED IN
環保在線 設計制作，未經允許翻錄必究 .? ? ?
請輸入賬號
請輸入密碼
請輸驗證碼
