Overview

  • Product name
  • Description
    Rabbit polyclonal to CTGF
  • Host species
    Rabbit
  • Specificity
    This antibody detects recombinant human CTGF by Western Blotting. The band can be blocked by the immunising peptide (ab13741). We have been unable to detect endogenous protein in WB with ab5097.
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IF, IHC-Frmore details
  • Species reactivity
    Reacts with: Rat, Human, Pig
  • Immunogen

    Synthetic peptide corresponding to Human CTGF aa 150-250 conjugated to keyhole limpet haemocyanin.
    Database link: P29279
    (Peptide available as ab13741)

  • Positive control
    • This antibody gave a positive signal in recombinant human CTGF in WB; This antibody gave a positive signal in the following Formaldehyde fixed cell lines:HepG2.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS. pH 7.4
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab5097 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
IHC-P Use at an assay dependent concentration. PubMed: 17028989
WB 1/500. Predicted molecular weight: 38 kDa.

This application has only been tested on recombinant protein.

ICC/IF Use a concentration of 5 µg/ml.
IHC-Fr 1/200.

Target

  • Function
    Major connective tissue mitoattractant secreted by vascular endothelial cells. Promotes proliferation and differentiation of chondrocytes. Mediates heparin- and divalent cation-dependent cell adhesion in many cell types including fibroblasts, myofibroblasts, endothelial and epithelial cells. Enhances fibroblast growth factor-induced DNA synthesis.
  • Tissue specificity
    Expressed in bone marrow and thymic cells. Also expressed one of two Wilms tumors tested.
  • Sequence similarities
    Belongs to the CCN family.
    Contains 1 CTCK (C-terminal cystine knot-like) domain.
    Contains 1 IGFBP N-terminal domain.
    Contains 1 TSP type-1 domain.
    Contains 1 VWFC domain.
  • Cellular localization
    Secreted > extracellular space > extracellular matrix. Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • CCN 2 antibody
    • CCN family member 2 antibody
    • CCN2 antibody
    • Connective tissue growth factor antibody
    • Ctgf antibody
    • CTGF_HUMAN antibody
    • Hcs 24 antibody
    • Hcs24 antibody
    • Hypertrophic chondrocyte specific protein 24 antibody
    • Hypertrophic chondrocyte-specific gene product 24 antibody
    • Hypertrophic chondrocyte-specific protein 24 antibody
    • IBP-8 antibody
    • IGF-binding protein 8 antibody
    • IGFBP-8 antibody
    • IGFBP8 antibody
    • Insulin like Growth Factor Binding Protein 8 antibody
    • Insulin-like growth factor-binding protein 8 antibody
    • MGC102839 antibody
    • NOV 2 antibody
    • NOV2 antibody
    see all

Images

  • ab5097 staining CTGF in human bronchus tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
    Tissue was fixed in formaldehyde and an antigen retrieval step was performed using EDTA pH9.0. Samples were then incubated with ab5097 at a 1/400 dilution for 20 minutes at 25°C. The secondary used was an undiluted HRP conjugated goat anti-mouse/rabbit polyclonal.

    See Abreview

  • ICC/IF image of ab5097 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab5097 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ab5097 staining CTGF in human heart tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent fixation in formaldehyde, heat mediated antigen retrieval in buffer and blocking (5 minutes/peroxidase block then 10 minutes/protein block) for 15 minutes at 20°C. The primary antibody was diluted, 1/50 and incubated with sample for 45 minutes at 20°C. A HRP conjugated goat polyclonal to rabbit IgG was used undiluted as secondary.

    See Abreview

  • ab5097 staining CTGF in Human Hela, cervix cancer cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS and blocked with 1% normal goat serum for 30 minutes. Samples were incubated with primary antibody (1/750) for 4 hours at 20°C. A Cy3®-conjugated Goat anti-rabbit polyclonal was used as the secondary antibody (1/800).

    See Abreview

  • ab5097 staining CTGF in Pig skin tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% BSA for 2 hours at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/200 in blocking buffer) for 24 hours at 4°C. A BHRP-conjugated Goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

    See Abreview

  • ab5097 staining CTGF in Pig skin tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with formaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% serum for 1 hour at 20°C. Samples were incubated with primary antibody (1/200 in PBS Tween 0.01% + donkey serum 1%) for 16 hours at 4°C. An Alexa Fluor®488-conjugated Donkey anti-rat IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • Western blot, on recombinant Human CTGF produced in E. coli, using ab5097 at 1/500.

    Not yet tested against endogenous CTGF

     Secondary antibody - anti-rabbit HRP (ab6721)

References

This product has been referenced in:
See all 12 Publications for this product

Customer reviews and Q&As

1-10 of 26 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Pig Tissue sections (skin)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer
Permeabilization
No
Specification
skin
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jun 10 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Pig Tissue sections (Ureter)
Specification
Ureter
Fixative
Paraformaldehyde
Permeabilization
Yes - 0,2 % Trition/PBS
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1%

Ms. Beate Thode

Verified customer

Submitted Feb 22 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Hela, Cervix Cancer Cells)
Specification
Hela, Cervix Cancer Cells
Fixative
Paraformaldehyde
Permeabilization
Yes - 0,2 Triton/PBS
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1%

Ms. Beate Thode

Verified customer

Submitted Feb 22 2013

Answer

Thank you for your reply and for your cooperation.

As agreed, I have issued a free of charge replacement with one unit of ab6328; the order number is ****.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Answer

En compensation de tous les problèmes rencontrés avec les anti-CTGF, j'aimerais vous offrir un avoir contre la commande originale du ab5097 ou un anticorps de remplacement de votre choix.

Merci de m'indiquer la résolution qui vous conviendrait le mieux.

Read More

Question

I finally managed to find some time to run a few more tests with the new antibodies against CTGF you sent me a few weeks ago (see power point file attached).

I used human dermal fibroblast cells that were stimulated or not by 10ng/ml TGFb1 for 24h as a positive control (CTGF is well known to be strongly increased by TGFb1 treatment)

I have also used two other protein ladders from different suppliers in parallel of my usual one to compare MW estimation and they gave the same result.



The new lot (GR42950-1) of the reference ab5097 gave exactly the same results than the previous (GR75927): a single strong band around 50-55kDa that is not varying with TGFb1 treatment.



The other reference ab6992 gives a different pattern.

I initially tried 1/5000 dilution as advised in the datasheet but the signal was weak. With longer exposure (and consequently a very strong background) the same band at 55kDa appears, so as two bands around 40 and 45kDa.

The second test with more protein loaded and lower antibody dilution (1/1000) exhibited the same pattern, bands at 55kDa and 45kDa not varying much upon TGFb1 treatment. But interestingly, there was a diffuse band around 40kDa only in the TGFb1 stimulated sample, bjut absent in the control lysate, which I guess is the specific one for CTGF.

However, you can see the WB stills displays high background (5% BSA blocking though), and the non specific band appears as strong, if not stronger, than the specific one. These non specific bands are present in the pig lysate (last line) whereas the 40kDa band is (I thought I had a positive staining in IHC-Fr for CTGF on pig skin frozen sections with ab5097, the epidermis was strongly positive but I also saw a diffuse positive staining in the healing wound bed in the dermis, where I was expecting the signal to be, but now I have doubts that ab5097 may not be specific in IHC-Fr either).

Are there other antibodies working on pig for IHC-Fr I could try to check?



I don’t know what else to change to get a cleaner blot on human lysates with ab6992. Would you have other advices? Could you let me know what were the exact conditions used to validate this ab6992 please? (especially transfer on nitrocellulose or PVDF?)



Thanks

Best regards

Read More
Answer

J'espère que vous allez bien.

J'aimerais vous informer que mes collègues du laboratoire ont effectué de nouveaux tests en Western Blot avec l'anti-CTGF référence ab5097.

Ils ont essayé différents lysats tissulaires et cellulaire, y compris celui que vous m'aviez suggéré - NIH 3T3 (Mouse embryonic fibroblast cell line) Whole cell lysate, TGF beta 1 treated. Malgré les différentes optimisations, il n'a pas été possible d'obtenir un bon résultat en Western Blot avec la protéine endogène, mais uniquement avec la protéine recombinante. La fiche technique a d'ailleurs été mise à jour avec cette information.

J'espère que ces informations vous seront utiles. N'hésitez pas à me contacter de nouveau si vous avez des questions ou commentaires au sujet de cet anticorps.


Monoclonal de lapin gratuit pour tout achat d'anticorps primaire, dans la limite des stocks disponibles. Notez le code "RABMAB-XBSMG" sur votre prochaine commande d’anticorps primaire. Pour plus d'informations, visitez le lien suivant: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

Thank you for your inquiry.

Unfortunately, we are not able to release the immunogen sequence for this particular antibody as it is proprietary information.

We aim to provide as much information as possible to our customers, so I am sorry that this has not been possible on this occasion.

Should you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for your message. We appreciate your valuable feedback in the Abreviews.

I can confirm that you will need to place your order using the testing discount code forthe free of charge antibody before 20th August.

I hope this information will be helpful. If you have any further questions, please do not hesitate to contact me.

Read More

Question

I ran a few more tests with the new antibodies against CTGF you sent me a few weeks ago (see power point file attached). I used human dermal fibroblast cells that were stimulated or not by 10ng/ml TGFb1 for 24h as a positive control (CTGF is well known to be strongly increased by TGFb1 treatment). I have also used two other protein ladders from different suppliers in parallel of my usual one to compare MW estimation and they gave the same result.

The new lot (GR42950-1) of ab5097 gave exactly the same results than the previous (GR75927): a single strong band around 50-55kDa that is not varying with TGFb1 treatment : not specific to CTGF obviously!!!
The other reference ab6992 gives a different pattern.
I initially tried 1/5000 dilution as advised in the datasheet but the signal was weak. With longer exposure (and consequently a very strong background) the same band at 55kDa appears, so as two bands around 40 and 45kDa.
The second test consisted of more protein loaded (25ug instead of 10) and lower antibody dilution (1/1000). It exhibited the same pattern, bands at 55kDa and 45kDa not varying much upon TGFb1 treatment. But interestingly, there was a diffuse band around 40kDa only in the TGFb1 stimulated sample, and absent in the control lysate, which I guessed was the specific one for CTGF.
However, you can see the WB stills displays high background (5% BSA blocking though), and the non specific band appears as strong, if not stronger, than the specific one. These bigger bands are also present in the pig skin lysate (last line) whereas the 40kDa band isn’t, showing instead at band around 34kDa: maybe a species-specific post-translational modification?

The last test I did was comparing the pattern from cell lysates to supernatant (as CTGF is a secreted growth factor!). I concentrated the media either with centricon columns with 30kDa cut-off or acetone precipitation. I got no signal at all on the supernatant samples. The cell lysate showed again an awful background and a multi-band pattern : 40, 50, 60 kDa, but also a weaker band around 36kDa (maybe 38kda, it was a gradient gel so not much separation) that is increased in the TGFb1 treated sample, so it looks like this maybe be the good one, suffering from additional non-specific reaction of the antibody.
The last thing I can try is to use nitrocellulose membrane instead of PVDF, that may help with the background… I’ll try to borrow some and try that next week, but I doubt I will be able to get rid of the un-specific bands…
I have just went through the datasheet of the two other anti-CTGF antibodies available on your website:
ab94939 gave a weak signal on the recombinant protein, and when tested on cell lysate, it shows a multi-band pattern similar to what I’ve got with ab6992. However, it is mentioned that “CTGF contains two glycosylation sites (SwissProt) which may explain the higher migrating band.” Maybe that could that explain these additional bands I detect with ab6994 (but on my sampels, these band still don’t vary upon TGFB treatment…)? (That would probably be useful to indicate this information on the other CTGF antibodies’datasheets.)
ab51704 has apparently only been tested on recombinant protein, but gives a nice strong signal.
I’d like to give them a last try if that’s OK with you, as I absolutely need to analyse this protein. Would that be possible to test them both?

Sorry about the massive reply, but I thought you may be interested in the results I’ve got.

Thanks
Best regards

Read More
Answer

Thank you very much for your email and the detailed information about your results.

I forwarded your data to our characterisation team who, since your last email, have started to investigate the problem. It has been decided to retest the products in liver, thymus, spleen and bone marrow lysates as this is where we might expect to find high expression of this protein. The experiments are currently in progress, I will let you know about the outcome as soon as the tests are done.

Thank you for your patience and sorry about the inconvenience caused. I hope I will be able to provide a quick and adequate resolution to this problem.

Do not hesitate to contact me again if you have any questions or comments.

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1-10 of 26 Abreviews or Q&A

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