Bioyears 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.  

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.   

Store all kit components at 4ºC until their expiration dates.