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Immunofluorescence analysis of Aggrecan was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Aggrecan (BC-3) Mouse Monoclonal Antibody (Product # MA3-16888) at 2 µg/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conj µgate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing localization in the extracellular matrix. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Bovine, Dog, Horse, Cat, Guinea pig, Human, Sheep, Pig, Rabbit, Rat|
|Published species reactivity||Bacteria, Zebrafish, Human, Mouse|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||Synthetic peptide corresponding to human aggrecan ARGSV neoepitope.|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||1:1000 - 1:5000|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Frozen) (IHC (F))||1:10 - 1:2000|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10 - 1:2000|
|Western Blot (WB)||1:500 - 1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
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.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Dynamic remodeling of the extra cellular matrix during zebrafish fin regeneration.
MA3-16888 was used in immunohistochemistry - frozen section to analyze extracellular matrix protein expression during caudal fin regeneration in zebrafish.
|Govindan J,Iovine MK||Gene expression patterns : GEP (19:21)||2015|
Proteomic profiling and functional characterization of early and late shoulder osteoarthritis.
MA3-16888 was used in western blot to establish comparative proteomic profiles of healthy shoulders and those in the early and late stages of osteoarthritis
|Wanner J,Subbaiah R,Skomorovska-Prokvolit Y,Shishani Y,Boilard E,Mohan S,Gillespie R,Miyagi M,Gobezie R||Arthritis research & therapy (15:null)||2013|
Lyme disease spirochaetes possess an aggrecan-binding protease with aggrecanase activity.
MA3-16888 was used in western blot to study a novel aggrecanase produced by the Lyme disease spirochaete during human infection and the pathogenic significance
|Russell TM,Johnson BJ||Molecular microbiology (90:228)||2013|
Effects of a glycogen synthase kinase-3β inhibitor (LiCl) on c-myc protein in intervertebral disc cells.
MA3-16888 was used in immunocytochemistry to investigate the regulation of c-myc Wnt signaling
|Hiyama A,Sakai D,Arai F,Nakajima D,Yokoyama K,Mochida J||Journal of cellular biochemistry (112:2974)||2011|
AGC1; AGCAN; aggrecan; aggrecan 1; aggrecan 1 (chondroitin sulfate proteoglycan 1, large aggregating proteoglycan, antigen identified by monoclonal antibody A0122); aggrecan core protein; aggrecan epidermal growth factor-like domain-1; aggrecan, structural proteoglycan of cartilage; cartilage aggregating proteoglycan; cartilage-specific proteoglycan core protein; Chondroitin sulfate proteoglycan 1; Chondroitin sulfate proteoglycan core protein 1; CSPCP; CSPG1; CSPGCP; large aggregating proteoglycan; large aggregating proteoglycan, antigen; MSK16; SEDK
ACAN; Agc; AGC1; AGCAN; BOS_20116; CSPCP; CSPG1; CSPGCP; MSK16; SEDK