Product Overview :
The AGE-RAGE in vitro Binding Assay Kit is primarily designed to screening inhibitors of AGE2 (glyceraldehyde-modified AGE)-sRAGE interaction (soluble RAGE) in vitro. In addition, this kit can be used for the rapid and sensitive evaluation of inhibitor candidates as well as characterization of AGE2-sRAGE interaction in vitro. The recombinant sRAGE used in this assay kit was specially designed for keeping the correct conformation to bind efficiently to AGE2-BSA but not to BSA, which are immobilized on the microplate surface. This technique allows us measuring the interaction of recombinant sRAGE to AGE2-BSA in a solid-phase assay system such as a conventional ELISA system.
Description :
Advanced glycation end products (AGEs) are important biochemical compounds found in diabetes and may be associated with inflammatory processes. In the vessel wall, AGEs are bound to specific receptors to modulate many cellular properties by activating several signaling pathways. One of these receptors is called "Receptor for Advanced Glycation End product" (RAGE). RAGE is a multi-ligand member of the immunoglobulin superfamily of cell surface molecules that is expressed in a variety of cell lines, including endothelial cells, smooth muscle cells, mononuclear phagocytes, pericytes, neurons, cardiac myocytes, mesangial cells and hepatocytes. RAGE interacts with different structures to transmit a signal into the cell and recognizes three-dimensional structures rather than specific amino acid sequences. Therefore, RAGE seems to fulfill the requirements of a pattern-recognition receptor. As a member of the immunoglobulin superfamily, it interacts with a diverse class of ligands, including AGEs, HMGB1 (also known as Amphoterin), amyloid β-peptide, amyloid A, leukocyte adhesion receptors, prions, Escherichia coli curli operons, β-sheet fibrils and several members of the S100 protein superfamily including S100/calgranulins. Thus RAGE may have potential involvement in several pathological processes
including inflammation, diabetes, Alzheimer's disease (AD), systemic amyloidosis and tumor growth. AGE-RAGE interaction enhances the expression of genes encoding cytokines, growth factors and adhesion molecules, and increases the classical acute phase proteins. Potential approaches to prevention and treatment of diabetes and its complications include inhibition of AGE formation, breakage of preformed AGE-proteins crosslink, blockade of AGE-RAGE interactions with RAGE competitors, antibody antagonists and RAGE specific metabolic inhibition. Inhibition of AGE-RAGE complex formation is able to suppress the levels of pro-inflammatory cytokines and growth factors and may be considered as a target for overcoming diabetic complications.
Characteristic :
The Research Products AGE-RAGE in vitro Binding Assay Kit is a semi-quantitative solid phase binding assay between recombinant His-tagged soluble RAGE (sRAGE) and glyceraldehyde-modified BSA (AGE2-BSA). Plates are pre-coated with AGE2-BSA, which is able to bind to recombinant His-tagged sRAGE. Horse radish peroxidase (HRP)-conjugated anti-His-tag monoclonal antibody specifically reacts with recombinant His-tagged sRAGE that is trapped with AGE2-BSA immobilized on the microplate well surface. The AGE-RAGE in vitro Binding Assay Kit may be used to characterize AGE-RAGE interaction as well as to screen inhibitors of AGE-RAGE interaction in vitro.
To perform the test, the recombinant His-tagged sRAGE is diluted in Reaction Buffer, pipetted into the wells and allowed to bind to AGE2-BSA immobilized on the wells. After wash the wells, the amount of bound recombinant His-tagged sRAGE is measured by binding it with HRP-conjugated anti-His-tag monoclonal antibody, which then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent). The color is quantitated by spectrophotometry and reflects the relative amount of recombinant His-tagged sRAGE that binds to AGE2-BSA on the wells.
For screening inhibitors of AGE-RAGE interaction in vitro, test compounds or the inhibitor candidates including a monoclonal antibody are added to appropriate amount of His-tagged sRAGE in the wells pre-coated with AGE2-BSA in a similar fashion as described above, followed by evaluation of inhibitory effect on AGE-RAGE interaction by measuring the amount of His-tagged sRAGE2 on the wells.
Applications :
1) Screening inhibitors of AGE-sRAGE interaction in vitro.
2) Characterization of inhibitor candidates of AGE2-sRAGE interaction in vitro.
3) Screening monoclonal antibodies that inhibit AGE2-sRAGE interaction in vitro.
4) Characterization of AGE2-sRAGE interaction in vitro.
Notes :
Stop Solution is a strong acid. Wear disposable gloves and eye protection when handling the solution.
Storage :
Upon receipt, store all other components at 4°C;
Don't expose reagents to excessive light.
All of the reagents included in the Research Product AGE-RAGE in vitro Binding Assay Kit have been tested for stability. Reagents should not be used beyond the stated expiration date.
Kit Components :
All samples should be assayed in duplicate. The following components are supplied and are sufficien for the one 96-well microtiter plate kit.
AGE2-BSA coated Microplate: One microplate supplied ready to use, with 96 wells (12 strips of 8-wells) in a foil, zip-lock bag with a desiccant pack. Wells are coated with AGE2-BSA (glyceraldehyde-modified BSA).
BSA coated Microplate: One microplate supplied ready to use, with 96 wells (12 strips of 8-wells) in foil, zip-lock bag with a desiccant pack for non-specific binding control. Wells are coated with BSA.
Recombinant His-tagged sRAGE: One vial containing 1600AU of lyophilized recombinant His-tagged human sRAGE.
Reaction Buffer: One bottle containing 50 mL of 1X buffer; used for for reconstitution and dilution of Recombinant His-tagged sRAGE, and binding reaction. Ready to use.
20X Inhibitor Control Compound: One vial containing 200uL of compound DP, which was discovered in commercial chemical library using this AGE-RAGE in vitro Binding Assay Kit. This compound DP may be irritating to the skin, eyes and upper respiratory tract and hazardous in case of ingestion.
HRP conjugated anti-His-tag monoclonal antibody: One bottle containing 30 mL of HRP (horseradish peroxidase) conjugated anti-His-tag monoclonal antibody. Ready to use.
10X Wash Buffer: One bottle containing 100 mL of 10X buffer containing Tween-20.
Substrate Reagent: One bottle containing 20 mL of the chromogenic substrate, tetra methylbenzidine (TMB). Ready to use.
Stop Solution: One bottle containing 20 mL of 1 N H2SO4. Ready to use.
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