Configuration-specificMonoclonalAntibodyBasedRab35ActivationAssayKitCatalogNumber：8280120assaysProductDescriptionTheRabfamilyofGTPasesregulateseukaryoticvesicularmembranetraffic

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Configuration-specific Monoclonal Antibody Based

Rab35 Activation Assay Kit

Catalog Number：82801

20 assays

Product Description

The Rab family of GTPases regulates eukaryotic vesicular membrane traffic. Rab35 is with both plasma

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.

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.

These anti- Rab35-GTP monoclonal antibodies can also be used to monitor the activation of Rab35 in

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.

Assay Principle

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

GTPγS loading Rab35 activation assays. Briefly, anti-active Rab35 mouse monoclonal antibody will be

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.

Kit Components

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

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

mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.

4. Anti- Rab35, Rabbit Polyclonal Antibody (Catalog No. 21078): 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 Rab35, whereas

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).

4. Add 5 µl of 100 X GDP (to 1 mM, final concentration) to the second tube (negative control).

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

concentration).

Assay Procedure

I. Active Rab35 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 Rab35 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 in

step 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- Rab35 polyclonal antibody, freshly diluted 1:50~1000

(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 4^oC 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 Rab35 Activation

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

(lane 2), and was blotted with anti-Rab35 polyclonal antibody(Cat. #21078). Input control is shown in

the bottom panel.

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