Aggrecanase Activity Assay Kit

$810.00

The Aggrecanase Activity Assay Kit measures activity of aggrecanases. It is used to screen and characterize aggrecanase inhibitors. It consists of two modules: Aggrecanase module and the ELISA module. In this Assay a recombinant fragment of human aggrecan interglobular domain (aggrecan-IGD) is first digested with aggrecanase (ADAMTS4). The proteolytic cleavage of the substrate releases an aggrecan peptide with the N-terminal sequence ARGSVIL (ARGSVIL-peptide). The ARGSVIL-peptide-s is then quantified with two monoclonal anti-peptide antibodies. The Eagle Biosciences Aggrecanase Activity Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: 30 510 111 Categories: , ,

Aggrecanase Activity Assay Kit

The Aggrecanase Activity Assay Kit is For Research Use Only

Size: 1×96 wells
Sensitivity: 12pM
Dynamic Range: 0.024 – 1.5 nM
Incubation Time: 3.5 hours
Sample Type: Cell culture supernatants
Sample Size: 5 µL

Control Included


Assay Background for Aggrecanase Activity Assay

Aggrecan is a large aggregating proteoglycan of articular cartilage. It is found also in aorta tissue, discs, tendon and in the perineuronal net. The aggrecan core protein consists of 2317 amino acids. Up to 130 glucosaminoglycan chains are attached to the core protein and the total molecular mass can reach 2.2 – 3.0 x 106 daltons. Within the aggrecan molecule 3 globular domains (G1, G2 and G3) can be distinguished. Domains G1 and G2 are connected by a rod-shaped polypeptide called interglobular domain (IGD), while the sequence between domains G2 and G3 contains attachment regions for keratan sulfate and chondroitin sulfate chains. Aggrecan interacts via the G1 domain with hyaluronan and link protein to form large aggregates. Such aggregates can contain up to 50 -100 aggrecan monomers noncovalently bound to a single hyaluronan chain through 2 link proteins. Aggrecan degradation is catalyzed by proteinases of the matrix metalloproteinase and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) families. Aggrecan-cleaving ADAMTS4 and ADAMTS5, named also aggrecanase 1 and aggrecanase 2, hydrolyze aggrecan at five different sites in vitro and in vivo. Four cleavage sites are located in the chondroitin sulfate-rich region between aggrecan globular domains G2 and G3 (sites E1667 – G1668, E1480 – G1481, E1771 – A1772, E1871 – L1872), while one site is placed in the rod-like polypeptide between globular domains G1 and G2 (E373 – A374). In experiments with isolated proteins additional cleavage sites in aggrecan were identified both for ADAMTS4 and ADAMTS5. A third proteinase of the ADAMTS family, ADAMTS1, also hydrolyses aggrecan at multiple sites including the unique site in the aggrecan interglobular domain.


Related Products

Aggrecanase Sensitive Activity Assay Kit
Universal Protease Activity ELISA Assay
HtrA1 ELISA Assay Kit

Additional Information

Assay Background


Aggrecan is a large aggregating proteoglycan of articular cartilage [1]. It is found also in aorta tissue, discs, tendon [1] and in the perineuronal net [2]. The aggrecan core protein consists of 2317 amino acids [3]. Up to 130 glucosaminoglycan chains are attached to the core protein and the total molecular mass can reach 2.2 – 3.0 x 106 daltons [4].

Assay Principle


Aggrecan-IGD (provided with this kit) is incubated with standard aggrecanase (ADAMTS4) and samples of unknown aggrecanase activity. In addition to aggrecanase, different concentrations of aggrecanase inhibitors can be added. The reaction is stopped by dilution with EDTA-containing buffer.

Typical Standard Curve


Manual

Product Manual