Product Description

Small GTPases are a super-family of cellular signaling regulators. The Ras-like small G proteins, RalA/B,

are important components of Ras signaling pathways, implicated in the initiation and maintenance of

tumorigenic transformation, as well as vesicle transport, apoptosis, transcription, cell migration, and cell

proliferation.

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

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

antibody that specifically recognizes Ral-GTP, but not Ral-GDP, and a RalB specific rabbit polyclonal

antibody. 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-Ral-GTP monoclonal antibodies can also be used to monitor the activation of Ral in cells and

in tissues by immunohistochemistry.

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

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

Assay Principle

NewEast Biosciences 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.

Kit Components

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. 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-RalB, Rabbit Polyclonal Antibody （Catalog No. 21033）: 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.