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

1. Anti active Ral, Mouse Monoclonal Antibody (Catalog No. 26913): One vial – 22 µL (1

    mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody

    specifically recognizes Ral-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. 30303): 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 RalA, Rabbit Polyclonal Antibody (Catalog No. 21034): One vial – 100 µL (1 mg/ml) in

    PBS, pH 7.4, contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30302): 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.