Anti-Aggrecan Antibody, MMP Cleaved, clone AF-28, Ascites Free

Aggrecan core protein (UniProt P16112; also known as Cartilage-specific proteoglycan core protein, Chondroitin sulfate proteoglycan 1, Chondroitin sulfate proteoglycan core protein 1, CSPCP) is encoded by the ACAN (also known as AGC1, CSPG1, MSK16, SEDK) gene (Gene ID 176) in human. Aggrecan is the major extracellular matrix (ECM) proteoglycan in articular cartilage that, together with type II collagen, provides the cartilage with its mechanical properties of reversible compressibility. Aggrecan consists of two globular domains (G1 and G2) and an IGD (interglobular domain) sequence in between, followed by an extended region before the third globular domain (G3). The region between G2 and G3 is heavily substituted with negatively charged sGAG (sulfated glycosaminoglycan) and is further subdivided into the KS (keratan sulfate), CS1 (chondroitin sulfate region 1) and CS2 regions. Degradation of cartilage ECM, including aggrecan, is a hallmark in arthritic diseases and in joint injuries. Multiple aggrecan cleavage sites have been characterized, including those in G1 domain (by cathepsin K), IGD (by aggrecanases, calpain, cathepsins B and K), KS (by calpain and MMPs), CS1 (by calpain and MMPs), and CS2 (by aggrecanases, calpain, cathepsin D and MMPs). In addition, different MMPs exhibit differential affinities toward aggrecan, the IGD IPEN-FFGV site is cut by MMPs 1–3, 7–9, 12–16, 19 and 20, the CS1 region in bovine aggrecan is cut at multiple GVED-I/(L)SGL sites by MMP-3, the end of the CS2 region in bovine aggrecan is cut at RPAE-ARLE by MMPs 2, 3, 7 and 12. Aggrecan proteolysis in the IGD results in decreased tissue water-holding capacity due to a loss of sGAG-containing chains. On the other hand, aggrecan proteolysis in the CS1 and CS2 regions is a natural turnover process in mature cartilage and does not affect cartilage function.

Clone AF-28 is a cleavage-site-specific monoclonal antibody that recognizes polypeptides with N-terminal 345-FFGVG sequence (numbering based on mature form) found at the N-terminal end of MMP-digested aggrecan fragments. By immunoblotting, clone AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase, and matrilysin, but failed to detect the G2 fragments by elastase, trypsin, or cathepsin B cleavage. Competition studies confirmed that clone AF-28 does not react with peptides containing internal FFGVG sequence.

Epitope: N-terminus FFGVG neoepitope sequence.

KLH-conjugated linear peptide (FFGVGGEED-C) corresponding to a.a. 361-369 of human aggrecan core protein.

Immunohistochemistry Analysis: A 1:50 dilution from a representative lot detected MMP-cleaved aggrecan in human cartilage tissue.
Immunohistochemistry Analysis: A representative lot detected MMP-cleaved aggrecan in paraffin-embedded tibia sections from both wild-type mice and mice with decreased MMP-13 expression (Zhou, X., et al. (2010). Proc. Natl. Acad. Sci. U. S. A. 107(29):12919-12924).
Western Blotting Analysis: A representative lot detected a higher level of MMP-generated FFGV fragments in osteoarthritis/osteoarthritic (OA) cartilage than in normal cartilage samples (Struglics, A., and Hansson, M. (2012). Biochem. J. 446(2):213-223).
Western Blotting Analysis: A representative lot detected MMPs-digested G2 fragment from recombinant G1-G2, but not undigested, elastase- or trypsin-digested fragments (Mercuri, F.A., et al. (1999). J. Biol. Chem. 274(45):32387-32395).
Western Blotting Analysis: A representative lot detected MMP-13-digested fragments from human, pig, bovine and rat, but not shark, aggrecan. Clone AF-28 did not detect undegraded aggrecan (Fosang, A.J., et al. (1996). FEBS Lett. 380(1-2):17-20).
Western Blotting Analysis: A representative lot detected in vitro generated aggrecan fragments by MMPs, as well as various aggrecan fragments in arthritis patients-derived synovial fluids (Fosang, A.J., et al. (1995). Biochem. J. 310( Pt 1):337-343).

Research Category
Cell Structure

Research Sub Category
ECM Proteins

This Anti-Aggrecan Antibody, MMP Cleaved, clone AF-28, Ascites Free is validated for use in Western Blotting, Immunohistochemistry (Paraffin) for the detection of Aggrecan.

Evaluated by Western Blotting in human chondrocyte tissue lysate.

Western Blotting Analysis: 0.5 µg/mL of this antibody detected MMP-cleaved aggrecan in 10 µg of human chondrocyte tissue lysate.

~200-260 kDa observed. Target fragment(s) appear larger than the calculated moleuclar weight(s) due to glycosylation. Uncharacterized band(s) may appear in some lysates.

Protein G purified.

Format: Purified

Purified mouse monoclonal IgG1κ antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Stable for 1 year at 2-8°C from date of receipt.

Concentration: Please refer to lot specific datasheet.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.