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Analysis Affinity of MR-Nbs1 by KinExA (CAT#: STEM-MB-0033-CJ)

Introduction

The ATM protein kinase plays a central role in signaling the presence of DNA double-strand breaks (DSBs) 3 in eukaryotic cells. Activation of ATM occurs very rapidly, within a few minutes of DNA break incidence, and is dependent on the Mre11/Rad50/Nbs1 (MRN) complex for its recruitment to sites of DNA damage. MRN also functions to promote a transition in ATM conformation from an inactive dimeric form to an active monomeric form, and increases the affinity of ATM for its substrates.The MRN complex is a large, multifunctional protein assembly that possesses endo-and exonucleolytic activity through the Mre11 component and ATP binding, hydrolysis, and adenylate kinase activity through the Rad50 component. Mre11/Rad50 (MR) complexes in prokaryotes and MRN complexes in eukaryotes are important for DNA double-strand break recognition and repair.




Principle

KinExA is a two-stage analysis system. In the first stage, a number of solutions are prepared, where one partner remains constant (constant binding partner, or CBP) and the other (titrant) is variable, usually serially diluted. As the titrant is added, the free CBP decreases and is analysed by a sophisticated and precise microfluorescence measurement device. The signal generated can be mathematically related to the affinity (KD) of the two molecules for each other, as well as the kinetic parameters of binding (kon) and dissociation (koff).

Applications

Immunology/Inflammation, Toxicology; Pharmacology

Procedure

1. preparation of the functionalized beads which will capture the analyte for measuremen.
2. preparation of a series of solutions consisting of a constant initial concentration of one component of the binary reaction and serial dilutions of the other reactant. The component that is kept constant is the constant binding partner (CBP) , and is the one which will be analyzed.
3. each reaction mixture is sampled and the fluorescence of free CBP bound to the capture beads is obtained for subsequent numerical analysis.

Materials

• Sample Type: Antibodies, Cells, Small Molecules, Proteins, DNA, Lipids, Serum, & More
• Equipment: Kinetic Exclusion Assay (KinExA)
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