Configuration-specificMonoclonalAntibodyBasedArl1ActivationAssayKitCatalogNumber：8290120assaysProductDescriptionArf-likeprotein1(Arl1)isamemberoftheArffamilyofregulatoryGTPases

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Configuration-specific Monoclonal Antibody Based

Arl1 Activation Assay Kit

Catalog Number： 82901

20 assays

Product Description

Arf-like protein 1 (Arl1) is a member of the Arf family of regulatory GTPases, within the Ras superfamily of GTPases, and with highly conserved orthologs throughout eukaryotes. Arl1 is essential for early embryonic development in Drosophila and in Caenorhabditis elegans. Arl1 is most similar in primary sequence, cellular location, and function (regulation of membrane traffic) to Arf1–6 and even shares several common binding partners. In addition to its function in membrane traffic at the Golgi/trans-Golgi network, there are reports indicating a possible role for Arl1 in ion homeostasis in yeast.

Currently there is no direct assay to measure the activation of Arl1 GTPases.

NewEast Biosciences Arl1 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Arl1-GTP, but not Arl1-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-Arl1-GTP monoclonal antibodies can also be used to monitor the activation of Arl1 in cells ａnd in tissues by immunohistochemistry.

NewEast Biosciences Arl1 Activation Assay Kit provides a simple and fast method to monitor the activation of Arl1. Each kit provides sufficient quantities to perform 20 assays.

Assay Principle

NewEast Biosciences Arl1 Activation Assay Kit bases on the configuration-specific anti-Arl1-GTP monoclonal antibody to measure the active Arl1-GTP levels, either from cell extracts or from in vitro GTPγS loading Arl1 activation assays. Briefly, anti-active Arl1 mouse monoclonal antibody will be incubated with cell lysates containing Arl1-GTP. The bound active Arl1 will then be pulled down by protein A/G agarose. The precipitated active Arl1 will be detected by immunoblot analysis using anti Arl1 rabbit polyclonal antibody or anti-Arl1 mouse monoclonal antibody.

Kit Components

1. Anti-active Arl1, Mouse Monoclonal Antibody (Catalog No. 26924): One vial – 22 µL (1mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide.

This antibody specifically recognizes Arl1-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, 750mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.

4. Anti-Arl1, Mouse monoclonal Antibody (Catalog No. 26056): 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 4^oC 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

? 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 thegenomic 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 mediate use, orsnap 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 Arl1, whereas in vitro GTPγS protein loading will activate nearly 90% of the Arl1.

1. Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 µg of purified Arl1
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 ntrol).

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,
finalconcentration).

Assay Procedure

I. Active Arl1 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 Arl1 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- Arl1 monoclonal antibody, freshly diluted 1:50~1000

(depending on the amount of Arl1 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-Mouse 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 Arl1 Activation　Assay Kit. One should use the data below for reference only.

Arl1 Activation Assay. Purified GST-tagged Arl1 proteins were immunoprecipitated with theanti-active Arl1 monoclonal antibody (Cat. # 26924) after treated with GDP (lane 1) or GTPγS (lane 2),ａnd was blotted with anti-Arl1 monoclonal antibody (Cat. # 26056). Arl1 protein input control is shown　in the bottom panel.

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