Overview

  • Product name
    Anti-Heparan Sulfate Proteoglycan 2 antibody [A7L6]
    See all Heparan Sulfate Proteoglycan 2 primary antibodies
  • Description
    Rat monoclonal [A7L6] to Heparan Sulfate Proteoglycan 2
  • Host species
    Rat
  • Specificity
    Recognizes domain IV of heparan sulphate proteoglycan 2 / perlecan. The reactivity is independent of the galactosaminoglycan moieties. Therefore, the epitope is not sensitive to heparitinase treatment.
  • Tested applications
    Suitable for: ICC, IHC-P, IHC-Fr, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Pig, Fish
  • Immunogen

    High molecular mass material derived from the Engelbreth-Holm-Swarm (EHS) tumor matrix containing laminin, entactin and HSPG. The antibody was raised against the total Heparan Sulphate Proteoglycan protein; no epitope mapping was ever performed.

  • General notes


    Proteoglycans are macromolecules consisting of a variety of core proteins with covalently attached one or several polysaccharide chains of the glycosaminoglycan type (heparan sulphate, heparin, chondroitin sulphate, dermatan sulphate or keratan sulphate). At least two forms of basement membrane heparan sulphate proteoglycan (HSPG) have been identified. One with a large core protein (> 400 kD) and one with a small core protein (30 kD). The large HSPG is probably the most abundant basement membrane proteoglycan. It is located predominantly in the lamina lucida, where it forms clustered aggregates and interacts with other basement membrane components to form the matrix. In addition, it also plays a critical role in attachment of cells to the basal membrane via integrin receptors.

Properties

Applications

Our Abpromise guarantee covers the use of ab2501 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 1/25 - 1/200. Use amplification with ABC (avidin biotin complex).
IHC-P Use at an assay dependent dilution. Perform enzymatic antigen retrieval before commencing with IHC staining protocol. Use amplification with ABC (avidin biotin complex).
IHC-Fr Use at an assay dependent dilution. PubMed: 16055495Use amplification with ABC (avidin biotin complex).
IP Use at an assay dependent dilution.
WB 1/100 - 1/1000.

Target

  • Function
    Integral component of basement membranes. Component of the glomerular basement membrane (GBM), responsible for the fixed negative electrostatic membrane charge, and which provides a barrier which is both size- and charge-selective. It serves as an attachment substrate for cells. Plays essential roles in vascularization. Critical for normal heart development and for regulating the vascular response to injury. Also required for avascular cartilage development.
    Endorepellin in an anti-angiogenic and anti-tumor peptide that inhibits endothelial cell migration, collagen-induced endothelial tube morphogenesis and blood vessel growth in the chorioallantoic membrane. Blocks endothelial cell adhesion to fibronectin and type I collagen. Anti-tumor agent in neovascularization. Interaction with its ligand, integrin alpha2/beta1, is required for the anti-angiogenic properties. Evokes a reduction in phosphorylation of receptor tyrosine kinases via alpha2/beta1 integrin-mediated activation of the tyrosine phosphatase, PTPN6.
    The LG3 peptide has anti-angiogenic properties that require binding of calcium ions for full activity.
  • Tissue specificity
    Found in the basement membranes.
  • Involvement in disease
    Defects in HSPG2 are the cause of Schwartz-Jampel syndrome (SJS1) [MIM:255800]; a rare autosomal recessive disorder characterized by permanent myotonia (prolonged failure of muscle relaxation) and skeletal dysplasia, resulting in reduced stature, kyphoscoliosis, bowing of the diaphyses and irregular epiphyses.
    Defects in HSPG2 are the cause of dyssegmental dysplasia Silverman-Handmaker type (DDSH) [MIM:224410]. The dyssegmental dysplasias are rare, autosomal recessive skeletal dysplasias with anisospondyly and micromelia. There are two recognized types: the severe, lethal DDSH and the milder Rolland-Desbuquois form. Individuals with DDSH also have a flat face, micrognathia, cleft palate and reduced joint mobility, and frequently have an encephalocoele. The endochondral growth plate is short, the calcospherites (which are spherical calcium-phosphorus crystals produced by hypertrophic chondrocytes) are unfused, and there is mucoid degeneration of the resting cartilage.
  • Sequence similarities
    Contains 4 EGF-like domains.
    Contains 22 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 11 laminin EGF-like domains.
    Contains 3 laminin G-like domains.
    Contains 3 laminin IV type A domains.
    Contains 4 LDL-receptor class A domains.
    Contains 1 SEA domain.
  • Post-translational
    modifications
    Proteolytic processing produces the C-terminal angiogenic peptide, endorepellin. This peptide can be further processed to produce the LG3 peptide.
    N- and O-glycosylated; contains three heparan sulfate chains. The LG3 peptide contains at least three and up to five potential O-glycosylation sites but no N-glycosylation.
  • Cellular localization
    Secreted > extracellular space > extracellular matrix > basement membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • Basement membrane specific heparan sulfate proteoglycan core protein antibody
    • Endorepellin (domain V region) antibody
    • Heparan sulfate proteoglycan of basement membrane antibody
    • HSPG 2 antibody
    • HSPG antibody
    • Hspg2 antibody
    • LG3 peptide antibody
    • Perlecan antibody
    • PGBM_HUMAN antibody
    • PLC antibody
    • Schwartz Jampel syndrome 1 (chondrodystrophic myotonia) antibody
    • SJA antibody
    • SJS antibody
    • SJS1 antibody
    see all

Images

  • IHC image on a frozen section of human colon showing strong reactivity in the extracellular matrix and basement membrane.

  • Immunohistochemistry on frozen section of Human kidney showing strong reactivity in the extracellular matrix and basement membrane.

References

This product has been referenced in:
  • Horsley H  et al. A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection. Sci Rep 8:1238 (2018). Read more (PubMed: 29352171) »
  • Brauchle E  et al. Biomechanical and biomolecular characterization of extracellular matrix structures in human colon carcinomas. Matrix Biol 68-69:180-193 (2018). IHC ; Human . Read more (PubMed: 29605717) »
See all 18 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Application
Immunohistochemistry (Resin sections)
Sample
Human Tissue sections (Human tonsil)
Specification
Human tonsil

Abcam user community

Verified customer

Submitted Dec 31 2013

Answer

Thank you for calling us yesterday and reporting the problems you have encountered in using the anti-Heparan Sulphate Proteoglycan (Large) antibody [A7L6](ab2501) in western blotting.

I am sorry to hear youhave beenexperiencing difficulties with thisproducts. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As discussed over the phone, I have attached aquestionnaire so thatI can gather further information regarding the samples tested and the protocol used with this antibody. OnceI havereceive the completed questionnaire,I will look at the protocol and see if there are any suggestionsI can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. In order to understand the problem fully I would appreciate it if you could send any images you have collected with the antibody, and any other antibody you have used with the samples.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

Read More

Answer

Vielen Dank für Ihre Anfrage.

Leider bieten wir keinen Marker an, der höher als 225 kDa läuft. Allerdings habe ich einen Marker bis 300kDa bei Fermentas gefunden:

http://www.fermentas.de/product_info.php?info=p1496&language=de

Da Sie ein sehr schweres Protein detektieren möchten, möchte ich Ihnen empfehlen, einen Ureapuffer zu verwenden. Ich habe Ihnen ein sehr interessantes Paper über die Löslichkeit von verschieden Proteinen hinsichtlich des Kompartiments und des Gewichtes an diese Email gehängt.

Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Benutzen Sie unsere Produkte? Schicken Sie uns einen Abreview. Verdienen Sie sich eine Belohnung!
https://www.abcam.com/abreviews

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Answer

Thank you for contacting us with your question and for your patience while I have looked into your enquiry.

Unfortunatelywe don't have any information regarding reactivity of this clone following herapanase treatment. According to the literature (see attached), the antibody recognizes an epitope in domain IV which is stable after heparinase III (heparitinase I) treatment.

I am sorry that we don't have more information about this epitope, but if you have any further questions or if there is anything else that we can do for you, please don't hesitate to ask.

Read More

Answer

Thank you for your enquiry. I am sorry to confirm that we are not able to provide this particular antibody BSA and glycerol free. I can suggest you may like to consider purifying the antibody in order to remove the BSA and glycerol. We do have the following antibody purification kits: ab102784  Antibody Purification kit (3 purifications) Click here (or use the following: https://www.abcam.com/index.html?datasheet=102784).         ab102783  Antibody Purification kit (1 purification) Click here (or use the following: https://www.abcam.com/index.html?datasheet=102783). I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.    

Read More

Answer

Thank you for your reply and providing more protocol information. I would recommend to incubate the antibody overnight at 4C as this will promote the antibody binding to the protein of interrest. If you still experience problems with the overnight incubation I would be prepared in this instance to offer you a refund/credit note even though the Abpromise of 90 days covering the antibody has expired, according to our records. I look forward to hearing how you get on with an overnight incubation,

Read More

Answer

Best to use lysate of mouse EHS tumor cells as cultured cells do not produce much of Heparan sulphate proteoglycan. Try to use 7.5% gel as the molecule is very large (the large core protein is 400 kDa).

Read More

Answer

Thank you for your enquiry. We would suggest applying protease digestion. For details on IHC and antigen retrieval, please click on the Resources section on the top panel of the Abcam homepage, you might find some useful information there. If you need anything further or any help then please let us know.

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Question

BATCH NUMBER 62320 DESCRIPTION OF THE PROBLEM No signal at all in any tissue tested (no background, no non-specific staining, either) SAMPLE Mouse, various tissues (liver, kidney, muscle, embryo etc.) PRIMARY ANTIBODY # ab2501-1; dilution 1/50, 1/100, 1/200 in phosphate buffered saline (PBS) with 1% bovine serum albumine; incubation overnight at +4?C after that, wash step (3 times with PBS) SECONDARY ANTIBODY Rabbit-anti-rat IgG (H+L) biotinylated, affinity purified, mouse adsorbed (Vector Laboratories, Burlingame); dilution 1/200 (2.5 ?g/ml) in PBS; incubation for 30 minutes at RT After that, wash step (3 times with PBS) DETECTION METHOD Avidin-Biotin-Complex-method (Vectastain ABC Kit - Elite; Vector Laboratories) with DAB as chromogene POSITIVE AND NEGATIVE CONTROLS USED Negative: no primary antibody Positive: not possible ANTIBODY STORAGE CONDITIONS Aliquoted, undiluted, at ?20?C FIXATION OF SAMPLE Formalin-fixed (10% Formalin, for about 24 hours), paraffin-embedded ANTIGEN RETRIEVAL Various methods tested: enzymatic (trypsin), heat (citrate buffer at +95?C), target retrieval solution (DAKO) PERMEABILIZATION STEP none BLOCKING CONDITIONS Blocking of endogenous peroxidase: methanol with 0.5% H2O2, for 30 minutes at RT Blocking serum: normal rabbit serum, 1:5 diluted, for 20 minutes at RT No wash step after blocking serum HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Antigen retrieval; dilution of primary antibody ADDITIONAL NOTES

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Answer

Thank you for forwarding the complaint on-line. I have read through the protocol which seems to be absolutely fine. Looking at the order record, we have sold several vials of this batch without any problem. The antibody should recognize Heparan Sulphate Proteoglycan in mesodermal tissues and organs. Therefore at this stage, I could either offer a new vial (we have the same batch in stock) or a reimbursement. Please do let us know how your customer would like to proceed.

Read More

Question
Answer

Thank you for your enquiry. All the information that we have regarding this antibody is located on the online datasheet. Formalin fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed; if no antigen retrieval step is stated on the antibody data sheet, we recommend to start off by trying the heat mediated pressure cooker method. If you have any more questions, please contact us again.

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1-10 of 13 Abreviews or Q&A

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