Configuration-specific Monoclonal Antibody Based

Sar1 Activation Assay Kit

Catalog Number：81801

20 assays

Product Description

    Small GTPases are a super-family of cellular signaling regulators. Sar1 is a member of these small

GTPases. Sar1 functions as a molecular switch to control protein–protein and protein–lipid

interactions that direct vesicle budding from the ER. Sar1 is invloved in membrane trafficking, and

regulates the assembly and disassembly of COPII coats.Currently there is no direct assay to measure the activation of Sar1 GTPases.

    NewEast Biosciences Sar1 Activation Assay Kit is based on the configuration-specific monoclonal

antibody that specifically recognizes Sar1-GTP, but not Sar1-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-Sar1-GTP monoclonal antibodies can also be used to monitor the activation of Sar1 in cellsand in tissues by immunohistochemistry.

    NewEast Biosciences Sar1 Activation Assay Kit provides a simple and fast method to monitor the

activation of Sar1. Each kit provides sufficient quantities to perform 20 assays.  

Assay Principle

    NewEast Biosciences Sar1 Activation Assay Kit bases on the configuration-specific anti-Sar1-GTP monoclonal antibody to measure the active Sar1-GTP levels, either from cell extracts or from in vitro GTPγS loading Sar1 activation assays. Briefly, anti-active Sar1 mouse monoclonal antibody will be incubated with cell lysates containing Sar1-GTP. The bound active Sar1 will then be pulled down by protein A/G agarose. The precipitated active Sar1 will be detected by immunoblot analysis using anti-Sar1 rabbit polyclonal antibody. 

Kit Components

1. Anti-active Sar1, Mouse Monoclonal Antibody (Catalog No. 26916): One vial – 22 ?L
    (1mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody

    specifically recognizes Sar1-GTP from all vertebrates. 

2. Protein A/G Agarose (Catalog No. 30301): One vial – 400 ?L of 50% slurry.

3. 5X Assay/Lysis Buffer (Catalog No. 30303): One bottle – 30 mL of 50 mM HEPES (pH8),
    0.1mg/ml BSA ,0.1%Triton-100, 1 mM DTT.

4. Anti-Sar1, Rabbit Polyclonal Antibody (Catalog No. 21090): One vial – 22 ?L (1 mg/ml) in

    PBS, pH 7.4, contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30302): One vial –100 ?l at 10 mM, use 5 ?L of GTPγS forGTP-
    labeling
    of 0.5 mL of cell lysate.

6. 100 X GDP (Catalog No. 30304): One vial –100 ?l at 100 mM, use 5 ?L of GDP for GDP-
    labeling  of 0.5 mL of cell lysate.  

Storage

Store all kit components at 4?C 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 ?g/mL leupeptin, and 10 ?g/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
    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 Sar1,
    whereas in vitro GTPγS protein loading will activate nearly 90% of the Sar1.

1, Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 ?g of purified
    Sar1 protein).

2, To each tube, add 20 ?l of 0.5 M EDTA (to 20 mM final concentration).

3, Add 5 ?l of 100 X GTPγS (to 100 ?M, final concentration) to one tube (positive control).

4, Add 5 ?l 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 ?l of 1 M MgCl2
     (to 60 mM, final concentration). 

Assay Procedure

I. Active Ran 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 ?l anti-active Sar1 monoclonal antibody to the tube.

4. Thoroughly resuspend the protein A/G Agarose bead slurry by vortexing or titurating.

5. Quickly add 20 ?L 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 ?L 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 ?L/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 100% 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-Sar1 rabbit polyclonal antibody, freshly diluted
    1:50~1000(depending on the amount of Sar1 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 temperaturewith 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 Sar1 Activation Assay Kit. One should use the data below for reference only. 

Sar1 activation assay. Purified Sar1 proteins were loaded with GDP (lanes 2 and 3) or GTPγS

(lanes 4 and 5). Immunoprecipitation was done with the anti-active Sar1 monoclonal antibody (Cat.No. 26916). Immunoblot was with an anti-Sar1 rabbit polyclonal antibody (Cat. No. 21090).