Overview

  • Product name
    Anti-Aggrecan ARGxx antibody [BC-3]
  • Description
    Mouse monoclonal [BC-3] to Aggrecan ARGxx
  • Host species
    Mouse
  • Specificity
    Recognises the aggrecanase (ADAMTS-1, -4 & -5)-generated N-terminal neoepitope ARG after cleavage between amino acids EGE and ARG within the interglobular domain of aggrecanase-catabolised aggrecan (Human aggrecan sequence enumeration). This antibody will not recognise the sequence ...ARG.. if it is in the non-cleaved intact aggrecan protein core; i.e. it will only recognise the aggrecanase generated neoepitope ARG... Samples need to be deglycosylated for epitope recognition (see notes).
  • Tested applications
    Suitable for: ICC/IF, ELISA, IHC-P, IHC-Fr, WB, Sandwich ELISAmore details
  • Species reactivity
    Reacts with: Rat, Sheep, Rabbit, Horse, Guinea pig, Cow, Cat, Dog, Human, Pig
    Does not react with: Mouse
  • Immunogen

    ARGSV... synthetic peptide conjugate.

  • General notes
    Samples must be deglycosylated using 0.01 Units Chondroitinase ABC (Sigma), 0.01 Units Keratanase (Seikagaku) and 0.0001 Units Keratanase II (Seikagaku) per 10µg S-GAG of non-deglycosylated aggrecan for optimal epitope recognition. See Little et al. and Caterson et al. Diluted Technomouse culture supernatant.


    Aggrecan degradation products containing this neoepitope are rapidly released from the tissue in model explant culture systems and are also present in the synovial fluids of patients with degenerative joint disease.

Properties

Applications

Our Abpromise guarantee covers the use of ab3773 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use at an assay dependent concentration. PubMed: 24312401
ELISA Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.

This antibody should work in IHC on formalin- or paraformaldehyde-fixed paraffin embedded sections.

IHC-Fr Use at an assay dependent concentration.

This antibody should work in IHC on alcohol-fixed frozen sections or un-fixed snap-frozen sections.

WB 1/100. Detects a band of approximately 50-250 kDa.

Detects a variety of epitopes between 50 and 250 kDa.

Sandwich ELISA Use at an assay dependent concentration. PubMed: 19932675

Target

Images

  • Immunocytochemistry/ Immunofluorescence analysis of AC cells labeling Aggrecan ARGxx with ab3773 at 1/200 dilution. Staining with the BC-3 antibody (red) that recognizes the neoepitope on human aggrecan that is created after cleavage within the sequence TEGE373-374ARGS by aggrecanases activity, decorated the cytoplasm of AC cells. The staining pattern was consistent with a vesicular distribution (arrows). For aggrecan detection, cells were treated postfixation with 5x10-5 U /µL of Chondroitinase ABC (Sigma) for 3 hours at 37°C. Nuclei are stained blue with Hoechst.

  • Immunocytochemistry/ Immunofluorescence analysis of NP cells labeling Aggrecan ARGxx with ab3773 at 1/200 dilution. Staining with the BC-3 antibody (red) that recognizes the neoepitope on human aggrecan that is created after cleavage within the sequence TEGE373-374ARGS by aggrecanases activity, decorated the cytoplasm of NP cells. The staining pattern was consistent with a vesicular distribution (arrows). For aggrecan detection, cells were treated postfixation with 5x10-5 U /µL of Chondroitinase ABC (Sigma) for 3 hours at 37°C. Nuclei are stained blue with Hoechst.

References

This product has been referenced in:
  • Shen L  et al. Overexpression of growth and differentiation factor-5 inhibits inflammatory factors released by intervertebral disc cells. Exp Ther Med 15:3603-3608 (2018). Read more (PubMed: 29545889) »
  • Choi H  et al. A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis. Matrix Biol N/A:N/A (2018). ICC/IF . Read more (PubMed: 29605718) »
See all 25 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Answer



Nicht aufgereinigte Antikörper wie Zellkulturüberstand, Vollserum oder Aszites haben keine determinierte Konzentration und können stark in Ihrer Konzentration schwanken.

Wenn die spezifische Antikörperkonzentration unbekannt ist, werfen Sie einen Blick auf die „Ungefähre Konzentration“ in unserer Tabelle in den FAQs, um den Wert abschätzen zu können. Bitte beachten Sie, dass diese Konzentrationen als Richtlinien benutzt werden sollten und die verwendeten Antikörperkonzentrationen daher Ihren Resultaten angepasst werden müssen.

Laut der Tabelle ist die ungefähre Immunglobulin- Konzentration 1 - 3 mg/ml.

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Question
Answer

TSC - was cultured in serum?
A: Serum was used during culture.

is the Ab available in purified form?
A: We do not routinely purify the antibody and so cannot give an answer to the second question. However, it could be purified.

I hope this is helpful. Please contact us again if you have any further questions.

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Answer

Thank you for your inquiry. Please find a list of possible positive controls below: ab2426: skin, lymph nodes Click here (or use the following: https://www.abcam.com/index.html?datasheet=2426). ab3773: cartilage samples Click here (or use the following: https://www.abcam.com/index.html?datasheet=3773). ab58632: Type X collagen is a product of hyperthrophic chondrotocytes and has been localized to presumptive mineralization zones of hyaline cartilage. Click here (or use the following: https://www.abcam.com/index.html?datasheet=58632). ab66025: The recommended positive control tissues are rat head tissue, rat embryo tissue, human osteosarcoma tissue, human glioma tissue, human intestinal cancer tissue. Click here (or use the following: https://www.abcam.com/index.html?datasheet=66025). ab102711: Specifically expressed in osteoblasts Click here (or use the following: https://www.abcam.com/index.html?datasheet=102711). I hope this information is helpful for your customer. I wish them good luck with their experiments.

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Question
Answer

I have been informed of the following information which I hope will help you: Ab3773: this should be reactive with goat since this antibody recognises a sequence ARG... that is conserved in all species Guinea Pig/rat to human but not mouse for some reason where the sequence is ALG.... This antibody will not recognise the sequence ...ARG.. if it is in the non-cleaved intact aggrecan protein core; i.e. it will only recognise the aggrecanase generated neoepitope ARG... Ab3778: this antibody recognises a linear sequence ..EPEEP... that is present in the IGD protein core of human and bovine aggrecan but not pig and several other animals; it seems it is not present in goat protein after BLASTing the sequence in goat. This antibody recognises the linear epitope ...EPEEP... in both intact and matrix protease degraded aggrecan from human and bovine cartilage. Please do not hesitate to contact me if I can be of further help,

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Answer

Thank you for your enquiry. The following reference gives details of the protocol used with this antibody in immunohistochemistry: Roberts S et al. Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. Spine 25:3005-13 (2000). PubMed: 11145811 I have added the relevant protocol to our online datasheet for your convenience and include below the following publications which have all used this product. This product has been used in: Karsdal MA et al. Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase-mediated cartilage degradation, but not aggrecanase-mediated cartilage degradation. Arthritis Rheum 56:1549-58 (2007). PubMed: 17469134 Little CB et al. Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation. Matrix Biol 21:271-88 (2002). PubMed: 12009333 Flannery CR et al. Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites. J Biol Chem 277:42775-80 (2002). PubMed: 12202483 Malfait AM et al. Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J Biol Chem 277:22201-8 (2002). PubMed: 11956193 Roberts S et al. Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. Spine 25:3005-13 (2000). PubMed: 11145811 Caterson B et al. Mechanisms involved in cartilage proteoglycan catabolism. Matrix Biol 19:333-44 (2000). PubMed: 10963994 Billington CJ et al. An aggrecan-degrading activity associated with chondrocyte membranes. Biochem J 336 ( Pt 1):207-12 (1998). PubMed: 9806902 Buttle DJ et al. "Aggrecanase" activity is implicated in tumour necrosis factor alpha mediated cartilage aggrecan breakdown but is not detected by an in vitro assay. Mol Pathol 50:153-9 (1997). PubMed: 9292151 Hughes CE et al. Monoclonal antibodies that specifically recognize neoepitope sequences generated by 'aggrecanase' and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro. Biochem J 305 ( Pt 3):799-804 (1995). PubMed: 7531436 This clone has been used in: Molecular Approaches to Osteoarthritis Research. Abstracts of the Kennedy Institute of Rheumatology Symposium, London, United Kingdom, April 18-20, 2004. Scand J Rheumatol Suppl :1-41 (2005). PubMed: 15864865 Roberts S et al. Matrix turnover in human cartilage repair tissue in autologous chondrocyte implantation. Arthritis Rheum 44:2586-98 (2001). PubMed: 11710715 Aplin JD et al. Human endometrial MUC1 carries keratan sulfate: characteristic glycoforms in the luminal epithelium at receptivity. Glycobiology 8:269-76 (1998). PubMed: 9451036

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ELISA
Sample
Human Recombinant protein (Part of aggrecan peptide 41AA)
Specification
Part of aggrecan peptide 41AA
Type
Indirect

Abcam user community

Verified customer

Submitted Nov 29 2006

Answer

Thank you for your patience. I can see that my colleague has also written to you and referred you to the paper in which the antibody was tested, i.e. Little CB et al., Matrix Biol 21:271-88 (2002). In summary the antibody detects a variety of epitopes between 50 and 250 kDa. I also contacted the originator of the antibody. Using ab3773 in Western blots can provide a wide range (of MW) of bands that are immuno-positive for the BC-3 neoepitope (i.e. ARG... as a result of IGD aggrecanase cleavage). Thus, if you are looking at media from cartilage explant cultures exposed to cytokines such as IL-1 you will find a wide range of MW product usually from 50kD - 250kD. However, if you use cartilage from young animals this can give mainly large MW bands and if the aggrecanase activity has been low you may see only bands at 40/50 - 90kD. I hope this additional information helps you.

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Question
Answer

The antibody ab3773 was tested in western blot analysis on proteoglycan metabolites released into the culture media and extracted from the cartilage matrix in cultures of bovine nasal cartilage maintained for 21 days in the paper Little CB et al. Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation. Matrix Biol 21:271-88 (2002). In figure 2 you will be able to see that the antibody detected bands of different sizes and thickness depending on the absence of 50 ng/ml interleukin-1 alone (IL-1), and IL-1 plus an inhibitor of MMPs at a high dose (1 mM) or a low dose (40 nM). In summary the antibody detects a variety of epitopes between 50 and 250 kDa. Because of copyright I am not able to forward the image of the figure to you or add it to the datasheet, but I hope you will be able to access the paper to see the banding pattern observed with the antibody. I hope this information will help you,

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Answer

Thank you for getting back in touch with me. I have been in touch with the originator of this antibody. They have provided me with some further details. They mention that both ab3773 & ab3776 are not usually used for IHC studies of articular cartilage because aggrecan catabolites with these (aggrecanase ab3773 or ab3776) generated neoepitopes are not retained within the cartilage; i.e. They are usually released into the synovial fluid or media if cultures are being examined. It may be possible that some ab3773 & ab3776 positive catabolites may be retained in tendon or intervertebral disc tissue; however, we have not examined this. If this was to be undertaken on these non-cartilagenous tissues a dilution of 1:100 should suffice. Regarding using ab3777 (anti ...PEN) on cartilage; a dilution of 1:100 (same as Western blots) should suffice. If they are looking for aggrecanase-generated neoepitope then they should use ab3775 (anti- ...EGE). I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for getting back to me and highlighting these details on our datasheet. I can now appreciate the deglycosylation that you have performed in line with the following publication. Little CB et al. Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation. Matrix Biol 21:271-88 (2002). PubMed: 12009333. I am sorry to heat that you have been having difficulties with this antibody. I would like to offer you credit against your initial purchase provided it was made within the past 90 days. If this is the case please e-mail me details of the order number and date of purchase.

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1-10 of 21 Abreviews or Q&A

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