Configuration-specific Monoclonal Antibody Based
Rab35 Activation Assay Kit
The Rab family of GTPases regulates eukaryotic vesicular membrane traffic. Rab35 is with both plasma
彩票app下载官网-正规彩票投注app-彩票app官方membrane and endosomal localization, and has been implicated in diverse processes that include T-cell receptor recycling, oocyte yolk protein recycling and cytokinesis. Rab35 regulates neurite outgrowth in neuronal-like cells, and can induce protrusions.
Currently there is no direct assay to measure the activation of Rab35 GTPases.
彩票app下载官网-正规彩票投注app-彩票app官方NewEast Biosciences Rab35 Activation Assay Kit is based on the configuration-specific monoclonal
antibody that specifically recognizes Rab35-GTP, but not Rab Rab35-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.
彩票app下载官网-正规彩票投注app-彩票app官方These anti- Rab35-GTP monoclonal antibodies can also be used to monitor the activation of Rab35 in
彩票app下载官网-正规彩票投注app-彩票app官方cells and in tissues by immunohistochemistry.
NewEast Biosciences Rab35 Activation Assay Kit provides a simple and fast method to monitor the
activation of Rab35. Each kit provides sufficient quantities to perform 20 assays.
NewEast Biosciences Rab35 Activation Assay Kit bases on the configuration-specific anti-Rab35-GTP
monoclonal antibody to measure the active Rab35-GTP levels, either from cell extracts or from in vitro
彩票app下载官网-正规彩票投注app-彩票app官方GTPγS loading Rab35 activation assays. Briefly, anti-active Rab35 mouse monoclonal antibody will be
彩票app下载官网-正规彩票投注app-彩票app官方incubated with cell lysates containing Rab35-GTP. The bound active Rab35 will then be pulled down
by protein A/G agarose. The precipitated active Rab35 will be detected by immunoblot analysis using
anti- Rab35 rabbit polyclonal antibody.
1. Anti-active Rab35, Mouse Monoclonal Antibody (Catalog No. 26922): One vial – 22 µL (1
mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody
彩票app下载官网-正规彩票投注app-彩票app官方 specifically recognizes Rab35 -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
彩票app下载官网-正规彩票投注app-彩票app官方 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
彩票app下载官网-正规彩票投注app-彩票app官方4. Anti- Rab35, Rabbit Polyclonal Antibody (Catalog No. 21078): One vial – 100 µL (1 mg/ml)
in PBS, pH 7.4, contained 50% glycerol.
彩票app下载官网-正规彩票投注app-彩票app官方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.
Store all kit components at 4ºC until their expiration dates.
Materials Needed but Not Supplied
1. Stimulated and non-stimulated cell lysates
彩票app下载官网-正规彩票投注app-彩票app官方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
彩票app下载官网-正规彩票投注app-彩票app官方8. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05%
彩票app下载官网-正规彩票投注app-彩票app官方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
彩票app下载官网-正规彩票投注app-彩票app官方• 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.
1. Culture cells (one 10-cm plate, ~ 107cells) to approximately 80-90% confluence. Stimulate
cells with activator or inhibitor as desired.
彩票app下载官网-正规彩票投注app-彩票app官方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
彩票app下载官网-正规彩票投注app-彩票app官方 cells (0.5- 1 mL per 10 cm tissue culture plate).
4. Place the culture plates on ice for 10-20 minutes.
彩票app下载官网-正规彩票投注app-彩票app官方5. Detach the cells from the plates by scraping with a cell scraper.
彩票app下载官网-正规彩票投注app-彩票app官方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 27½-gauge syringe needle 3-4 times to shear the
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.
彩票app下载官网-正规彩票投注app-彩票app官方1. Culture cells and stimulate with activator or inhibitor as desired.
2. Perform a cell count, and then pellet the cells by centrifugation.
彩票app下载官网-正规彩票投注app-彩票app官方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.
彩票app下载官网-正规彩票投注app-彩票app官方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 27½-gauge syringe needle 3-4 times to shear the
彩票app下载官网-正规彩票投注app-彩票app官方 genomic DNA.
8. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).
彩票app下载官网-正规彩票投注app-彩票app官方9. Collect the supernatant and store samples on ice for immediate use, or snap freeze and store at -
彩票app下载官网-正规彩票投注app-彩票app官方 70 °C for future use.
In vitro GTPγS/GDP Protein Loading for positive and negative controls
彩票app下载官网-正规彩票投注app-彩票app官方 Note: In vivo stimulation of cells will activate approximately 10% of the available Rab35, whereas
彩票app下载官网-正规彩票投注app-彩票app官方 in vitro GTPγS protein loading will activate nearly 90% of the Rab35.
1. Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 µg of purified Rab35 protein).
2. To each tube, add 20 µl of 0.5 M EDTA (to 20 mM final concentration).
3. Add 5 µl of 100 X GTPγS (to 100 µM, final concentration) to one tube (positive control).
彩票app下载官网-正规彩票投注app-彩票app官方4. Add 5 µl of 100 X GDP (to 1 mM, final concentration) to the second tube (negative control).
彩票app下载官网-正规彩票投注app-彩票app官方5. Incubate the 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 (to 60 mM, final
I. Active Rab35 Pull-Down Assay
彩票app下载官网-正规彩票投注app-彩票app官方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.
彩票app下载官网-正规彩票投注app-彩票app官方3. Add 1 µl anti-active Rab35 monoclonal antibody to the tube.
彩票app下载官网-正规彩票投注app-彩票app官方4. Thoroughly resuspend the protein A/G Agarose bead slurry by vortexing or titurating.
彩票app下载官网-正规彩票投注app-彩票app官方5. Quickly add 20 µL of resuspended bead slurry to each tube.
彩票app下载官网-正规彩票投注app-彩票app官方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
10. After the last wash, pellet the beads and carefully remove all the supernatant.
彩票app下载官网-正规彩票投注app-彩票app官方11. Resuspend the bead pellet in 20 µL of 2X reducing SDS-PAGE sample buffer.
12. Boil each sample for 5 minutes.
彩票app下载官网-正规彩票投注app-彩票app官方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
彩票app下载官网-正规彩票投注app-彩票app官方 recommended to include a pre-stained MW standard (as an indicator of a successful transfer in
彩票app下载官网-正规彩票投注app-彩票app官方2. Perform SDS-PAGE following the manufacturer’s instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s
III. Immunoblotting and Detection (all steps are at room temperature, with agitation)
彩票app下载官网-正规彩票投注app-彩票app官方1. Following the electroblotting step, immerse the PVDF membrane in 100% Methanol for 15
彩票app下载官网-正规彩票投注app-彩票app官方 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.
彩票app下载官网-正规彩票投注app-彩票app官方2. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature
with constant agitation.
彩票app下载官网-正规彩票投注app-彩票app官方 Incubate the membrane with anti- Rab35 polyclonal antibody, freshly diluted 1:50~1000
彩票app下载官网-正规彩票投注app-彩票app官方 (depending on the amount of Rab35 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 4o彩票app下载官网-正规彩票投注app-彩票app官方C 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
彩票app下载官网-正规彩票投注app-彩票app官方 with constant agitation.
5. Wash the blotted membrane three times with TBST, 5 minutes each time.
彩票app下载官网-正规彩票投注app-彩票app官方6. Use the detection method of your choice such as ECL.
Example of Results
The following figure demonstrates typical results seen with NewEast Biosciences Rab35 Activation
彩票app下载官网-正规彩票投注app-彩票app官方Assay Kit. One should use the data below for reference only.
Rab35 activation assay. Purified GST-tagged Rab35 proteins (Cat. #10132) were immunoprecipitated
with the anti-active Rab35 monoclonal antibody (Cat. #26922) after treated with GDP (lane 1) or GTPγS
彩票app下载官网-正规彩票投注app-彩票app官方(lane 2), and was blotted with anti-Rab35 polyclonal antibody(Cat. #21078). Input control is shown in
the bottom panel.
Hepatology Volume 61, Issue 1, pages 361–374, January 2015
Endocrinology. 2014 Sep;155(9):3315-28