Overview

  • Product name
    Anti-SOX9 antibody - ChIP Grade
    See all SOX9 primary antibodies
  • Description
    Rabbit polyclonal to SOX9 - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    The antibodies were evaluated for specificity with a dot blot assay using synthetically prepared SOX9 peptides. By immunoblot analysis, a ~65 kDa immunoreactive protein from mouse embryo cell lysate was detected (sample were boiled prior to subject 10% SDS-polyacrylamide gel electrophoresis). This band is abolished by the depletion of the synthetic peptide.
  • Tested applications
    Suitable for: IHC-Fr, ICC/IF, WB, IP, ELISA, IHC-P, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chicken, Cow
  • Immunogen

    Synthetic peptide:

    KYQPRRRKSVKNG

    , corresponding to amino acids 173 - 185 of Human/ Mouse/ Bovine SOX 9.

  • Positive control
    • 3T3 cells FFPE mouse e14 whole foetus tissue sections

Applications

Our Abpromise guarantee covers the use of ab3697 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-Fr 1/200. PubMed: 19388005
ICC/IF Use at an assay dependent concentration.
WB Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 62 kDa (predicted molecular weight: 56 kDa).
IP Use 3-5µg for 107 cells.
ELISA Use a concentration of 0.1 - 1 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ChIP Use at an assay dependent concentration. PubMed: 21872590

Target

  • Function
    Plays an important role in the normal skeletal development. May regulate the expression of other genes involved in chondrogenesis by acting as a transcription factor for these genes.
  • Involvement in disease
    Defects in SOX9 are the cause of campomelic dysplasia (CMD1) [MIM:114290]. CMD1 is a rare, often lethal, dominantly inherited, congenital osteochondrodysplasia, associated with male-to-female autosomal sex reversal in two-thirds of the affected karyotypic males. A disease of the newborn characterized by congenital bowing and angulation of long bones, unusually small scapulae, deformed pelvis and spine and a missing pair of ribs. Craniofacial defects such as cleft palate, micrognatia, flat face and hypertelorism are common. Various defects of the ear are often evident, affecting the cochlea, malleus incus, stapes and tympanum. Most patients die soon after birth due to respiratory distress which has been attributed to hypoplasia of the tracheobronchial cartilage and small thoracic cage.
  • Sequence similarities
    Contains 1 HMG box DNA-binding domain.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • campomelic dysplasia autosomal sex reversal antibody
    • CMD 1 antibody
    • CMD1 antibody
    • CMPD 1 antibody
    • CMPD1 antibody
    • SOX 9 antibody
    • Sox9 antibody
    • SOX9_HUMAN antibody
    • SRA 1 antibody
    • SRA1 antibody
    • SRXX2 antibody
    • SRXY10 antibody
    • SRY (sex determining region Y) box 9 (campomelic dysplasia autosomal antibody
    • SRY (sex determining region Y) box 9 antibody
    • SRY (sex determining region Y)-box 9 antibody
    • SRY (sex-determining region Y)-box 9 protein antibody
    • SRY related HMG box gene 9 antibody
    • Transcription factor SOX 9 antibody
    • Transcription factor SOX-9 antibody
    • transcription factor SOX9 antibody
    see all

Images

  • ChIP analysis of 15P-1 cells using no antibody (lane 1), a control rabbit IgG (lane 2) or ab3697 (lane 3). DNA from the Er71 promoter (-334 to +28), an Er71 region ranging from exon 6 to exon 7, or the 3'-UTR of Sox9 (2664–3023) was PCR amplified.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human skin hair follicles labling SOX9 with ab3697. Hematoxylin purple nuclear conterstain.

  • IHC-P image of SOX9 (ab3697) on E11.5 mouse embryo sections. The sections were fixed in paraformaldehyde and underwent heat mediated antigen retrieval using Tris/EGTA (pH9). The sections were then blocked in 1% BSA solution for 30 mins at 20°C. In these images, ab3697 can be seen staining nuclei on the lumen surface of the brain (green).

    See Abreview

  • Rabbit polyclonal antibody to Sox9 recognizes full length of Sox9 (left lane) and C-terminal deletion of Sox9 (right lane)

    (Compared to anti-Sox9 antibody from a different company - not shown).

    This Western blot image was kindly supplied as part of the review submitted by Lilia Topol.

  • Immunofluroescence analysis of murine nucleus pulposus (NP) cells, staining SOX9 with ab3697.
    Left panel = 2 day vehicle treated control
    Right panel = 2 day cyclopamine treated cells
  • IHC image of SOX9 staining in mouse e14 whole foetus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3697, 1µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • Immunofluroescence analysis of murine nucleus pulposus (NP) cells, staining SOX9 with ab3697.
    Left panel = control Shh flox mouse
    Right panel = Shh mutant mouse

References

This product has been referenced in:
  • Ashraf S  et al. Role of RHEB in Regulating Differentiation Fate of Mesenchymal Stem Cells for Cartilage and Bone Regeneration. Int J Mol Sci 18:N/A (2017). IHC-P . Read more (PubMed: 28441755) »
  • Wa Q  et al. miR-30b regulates chondrogenic differentiation of mouse embryo-derived stem cells by targeting SOX9. Exp Ther Med 14:6131-6137 (2017). Read more (PubMed: 29285169) »
See all 38 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Vascular smooth muscle cells)
Permeabilization
Yes - NP40
Specification
Vascular smooth muscle cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 31 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
CyTOF
Sample
Mouse Cell (lung)
Specification
lung

Irit Milman

Verified customer

Submitted Feb 06 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Human Chondrocyte cell line)
Specification
Human Chondrocyte cell line
Blocking step
BSA as blocking agent for 15 minute(s) · Concentration: 1000µg/mL · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 09 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 1.5% · Temperature: 25°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer, pH 6.1
Sample
Zebrafish Tissue sections (Juvenile zebrafish head including gills)
Specification
Juvenile zebrafish head including gills
Permeabilization
Yes - 70% Methanol/3% H2O2, room temp x 20 min
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 20 2014

Answer

We do not have any EMSA data for this SOX9 antibody, and we are not aware of any references or feedback from customers who have used it for EMSA. There are two references listed on the datasheet that have used it for ChIP. These might help convince you to try it for ChIP, without first trying EMSA.

Ditacchio L et al. Transcription Factors ER71/ETV2 and SOX9 Participate in a Positive Feedback Loop in Fetal and Adult Mouse Testis. J Biol Chem 287:23657-66 (2012).

Yang J et al. MiR-140 is co-expressed with Wwp2-C transcript and activated by Sox9 to target Sp1 in maintaining the chondrocyte proliferation. FEBS Lett 585:2992-7 (2011).

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Answer

I do think IP is a good idea, though you will see some strong signal at 50 and 25 kDa, if your secondary anti-rabbit secondary IgG detects denatured rabbit IgG, which will be in the IP product. It may not be a problem if the band of interest is near 56-60 kDa, which I am assuming is where you are looking for it. If you are interested, we have a product range called VeriBlot that addresses the problem of detection of IP antibody in the blot, for example anti-rabbit IgG ab131366:

Click here (or use the following: https://www.abcam.com/index.html?datasheet=131366).

Let me know if you have questions. Please be aware that ab71762 does not recognize SOX9 protein phosphorylated at S181 as stated in the specificity field of the datasheet, so that might account for diminished signal compared to ab3697 if the cells, differentiated or undifferentiated, are phosphorylated at S181.

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Answer

Thank you for contacting us.

I have checked our database and the immunogen sequence of ab3697 is indeed xxxxxxxx, corresponding to amino acids xxxxxx of Human / Mouse / Bovine SOX 9.

I have checked the sequence homology between human and the predicted rabbit sequence and there is 90.7% sequence homology for the full protein and 100% sequence similarity with the immunogen (see attachments) so it is likely that this antibody will detect rabbit SOX9.

However, since ab3697 is a rabbit polyclonal, it is likely that your secondary antibody won't only detect this antibody but also all other rabbit antibodies in your sample. You could therefore use one of our kits to directly conjugate ab3697 to a label e.g FITC, HRP, biotin. See the website below:

https://www.abcam.com/easylink

Alternatively, I can suggest you use a different antibody which is not a rabbit polyclonal. I suggest using ab76997 which is a mouse monoclonal (https://www.abcam.com/ab76997). Its immunogen is xxxxxxxxxxxxxxxxxcorresponding to amino acids xxxxxxxxxxxx of human SOX9 (NP_000337) with a 26 kDa tag. I have done a sequence alignment and have found 88% sequence homology therefore there are chances that this antibody will detect sox9 in rabbit 9 see attachment).


However, to our knowledge, ab76997 has not been tested in rabbit or IHC-Fr . However by participating in our AbTrial program you can now use our products in an untested application or species without financial risk.

Simply follow these easy steps below to apply for our AbTrial Program:

1. Reply to this email, letting us know you are interested in testing this product.

2. Our scientists will email you an inactive personal discount code for the value of the product.

3. Purchase and test the product at the regular price.

4. Submit your results, including your discount code in the additional notes section of your Abreview.

5. Once the Abreview is submitted, the discount code will become active.

6. Apply your discount code on your next order to receive that value off.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/abtrial.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (E11.5 embryo)
Specification
E11.5 embryo
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/EGTA pH=9
Permeabilization
No
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Aug 07 2012

Answer

Thank you for contacting us with your question.

Ab26414 works well with formalin-fixed, paraffin-embedded sections but it has not been sucessfully used with cryosections, so I wouldn't recommend using it in IHC-Fr.

Ab3697 has been tested with frozen tissue sectionsaccording toa journal article (attached to this email, see page 33 for an image) but the tissue was fixed with methanol. We haven't tested ab3697 with PFA-fixed tissue, but it is possible that it will work. If the tissue is fixed with PFA for more than 15-20 minutes, then I would recommend a heat-mediated antigen retrieval step.

I hope this information will be useful, but please let me know if you have any further questions or if there is anything else that we can do for you and I'll be happy to help.

Read More

Answer

I appreciate the time taken to submit further information to us.
Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results.
I apologise for the inconvenience and am pleased to offer you either a free of charge replacement for a new vial (unfortunately there are no different lots available), a different anti- SOX9 antibody, or a credit note instead. See below the link to the different alternatives suitable for IHC-P / IHC-Fr:
https://www.abcam.com/index.html?pageconfig=searchresults&search=SOX9&pt=1&sk=app&sv=78&sn=IHC-Fr&l=3&fViewMore=1
Please let me know which of the alternatives offered would be more suitable for you and I will be happy to help.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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1-10 of 18 Abreviews or Q&A

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