81401 Rap1 Activation Assay Kit

 Product Description

Small GTPases are a super-family of cellular signaling regulators. The small Ras-like GTPase Rap1 is an

evolutionary conserved protein that originally gained interest because of its capacity to revert the

morphological phenotype of Ras-transformed fibroblasts. Rap1 is regulated by a large number of stimuli

that include growth factors and cytokines, but also physical force and osmotic stress. Rap1 was shown to

regulate multiple basic cellular processes. The best studied aspect of Rap1 function in endothelial cells

involved its role in regulation of cell-cell junction formation and remodeling.

Currently there is no direct assay to measure the activation of Rap1 GTPases.

NewEast Biosciences Rap1 Activation Assay Kit is based on the configuration-specific monoclonal

antibody that specifically recognizes Rap1-GTP, but not Rap1-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-Rap1-GTP monoclonal antibodies can also be used to monitor the activation of Rap1 in cells

and in tissues by immunohistochemistry.

NewEast Biosciences Rap1 Activation Assay Kit provides a simple and fast method to monitor the

activation of Rap1. Each kit provides sufficient quantities to perform 20 assays.

Assay Principle

NewEast Biosciences bases on the configuration-specific anti-Rap1-GTP

monoclonal antibody to measure the active Rap1-GTP levels, either from cell extracts or from in vitro

GTPγS loading Rap1 activation assays. Briefly, anti-active Rap1 mouse monoclonal antibody will be

incubated with cell lysates containing Rap1-GTP. The bound active Rap1 will then be pulled down by

protein A/G agarose. The precipitated active Rap1 will be detected by immunoblot analysis using anti-

Rap1 rabbit polyclonal antibody.

Kit Components

1. Anti-active Rap1, Mouse Monoclonal Antibody （Catalog No. 26912）: One vial – 22 μL （1

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

specifically recognizes Rap1-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-Rap1, Rabbit Polyclonal Antibody （Catalog No. 21104）: 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.