* Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own experiment using appropriate negative and positive controls.
1mg/ml BSA, 30% glycerol
0.05% sodium azide
Product Specific Information
Purified recombinant Aggrecan Interglobular Domain (IGD) protein (Amino acids T331-G458 with C-term His-tag, expressed in E. coli), was used to validate this antibody. The recombinant protein contains cleavage sites for aggrecanases (E373-A374 in aggrecan) and matrix metalloproteinases (N341-F342 in aggrecan). The recombinant protein was cleaved in vitro using recombinant Aggrecanase. Degradation of IGD and accumulation of cleaved fragments was visualized via MEMCode reversible protein staining, followed by Western Blot using the BC-3 antibody for the specific detection of the aggrecan neoepitope. The antibody will also recognize full length IGD, as shown by ELISA, however with significantly lower affinity.
MA3-16888 has successfully been used in ELISA, immunohistochemistry (frozen and FFPE) and Western blot applications. This antibody may be used to immunostain formalin-fixed, paraffin-embedded sections, as well as alcohol-fixed frozen sections and un-fixed snap frozen sections. Samples must be deglycosylated using 0.01 Units Chondroitinase ABC, 0.01 Units Keratanase and 0.0001 Units Keratanase II per 10 µg S-GAG of non-deglycosylated aggrecan for optimal epitope recognition.
MA3-16888 detects aggrecan in human, bovine, feline, canine, guinea pig, equine, porcine, rabbit, rat, and sheep samples. Does not react with mouse samples.
Aggrecan also known as cartilage-specific proteoglycan core protein (CSPCP) or chondroitin sulfate proteoglycan 1 is a protein that is encoded by the ACAN gene. The encoded protein is an integral part of the extracellular matrix in cartilaginous tissue and it withstands compression in cartilage. The human form of the protein is 2316 amino acids long (~ 250 kDa) and can be expressed in multiple isoforms due to alternative splicing. Aggrecan exhibits a bottlebrush structure, in which chondroitin sulfate and keratan sulfate chains are attached to an extended protein core. The synthesis and degradation of aggrecan are being investigated for their roles in cartilage deterioration during joint injury, disease, and aging. It contains three interglobular domains (IGDs), G1, G2, and G3 that are involved in aggregation, hyaluronan binding, cell adhesion, and chondrocyte apoptosis. Aggrecan fragments from articular cartilage are released into the synovial fluid at all stages of human osteoarthritis as a result of cleavage by aggrecanase and MMP activity. Antibodies BC-3 and BC-4 can be used to detect these neo epitopes of Aggrecanase (ARGSVXXX) and MMP (XXXVDIPEN), respectively.
Aggrecan Antibody (MA3-16888) in WB Aggrecan-BC-3_Antibody_MA3-16888_Western-Blot_1_20130614063247.jpg
Western Blot with anti-Aggrecan Monoclonal Antibody [BC-3] (MA3-16888)
Western blot analysis of Aggrecan neoepitope fragments, generated as a result of Aggrecanase proteolytic activity, was performed using cleaved and uncleaved recombinant Interglobular domain (IGD) of Aggrecan (Product # RP-77527), followed by detection with an Aggrecan monoclonal antibody (Product # MA3-16888). IGD (5ug) was incubated for the indicated times with or without 1U recombinant Aggrecanase. IGD samples and 10ul of PageRuler Prestained Protein Ladder (Product # 26616) were loaded onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and full length IGD, as well as cleaved Aggrecan-IGD fragments were visualized (bottom panel) using a Reversible Protein Stain Kit for PVDF Membranes (Product # 24585). For specific neo-epitope fragment detection (top panel), the membrane was incubated in Destain solution (included in the kit), blocked with 5% BSA/TBST for 1 hour, and probed with an Aggrecan monoclonal antibody (Product # MA3-16888) at a dilution of 1:1000 overnight at 4°C on a rocking platform. The membrane was washed in TBS-0.1% Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
Aggrecan Antibody (MA3-16888) in ELISA Aggrecan-BC-3_Antibody_MA3-16888_ELISA_1_20130614063411.jpg
ELISA with anti-Aggrecan Monoclonal Antibody [BC-3] (MA3-16888)
ELISA analysis of Aggrecan was performed by coating Amine-binding Maleic Anhydride Activated Plates (Product # 15110) overnight with 100 µL of 2 µg/mL Aggrecan Interglobular Domain recombinant protein (Product # RP-77527). Plates were blocked for 1 hour at room temperature with StartingBlock Blocking Buffer (Product # 37538) and incubated with 100 µL per well of an Aggrecan monoclonal antibody (Product # MA3-16888) in duplicate at 25, 12.5, 6.25, 3.125, 1.563, 0.78 µg/100 µL with serial dilutions of for 2 hours at room temperature. The plate was washed and incubated with 100 µL per well of goat anti-mouse IgG-HRP secondary antibody (Product # 31430) in all test wells at 1:30,000 for 1 hour at room temperature. Detection was performed using 1-Step Ultra TMB - ELISA Substrate (Product # 34028) for 30 minutes at room temperature in the dark. The plate was then stopped with 0.16M sulfuric acid. Absorbance values were read on a spectrophotometer at 450-550 nm.
MA3-16888 was used in immunocytochemistry to investigate the regulation of c-myc Wnt signaling
J Cell Biochem. 2011 Oct;112(10):2974-86. "Effects of a glycogen synthase kinase-3? inhibitor (LiCl) on c-myc protein in intervertebral disc cells."
Author(s): Hiyama A, Sakai D, Arai F, Nakajima D, Yokoyama K, Mochida J
Number of Citations: 1
(See PubMed article )
(This product is for In Vitro experimental use only. Not for resale without express authorization.)