Overview

  • Product name

  • Description

    Rabbit polyclonal to Fibronectin
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ELISA, RIA, ICC/IF, IHC-Fr, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Immunogen

    Purified fibronectin from murine plasma.

  • Positive control

    • ICC/IF: Normal and cancer-associated fibroblasts; Mouse bone marrow cells. IHC-Fr: Mouse liver tissue. IHC-P: Human aorta tissue; Rat skin tissue.
  • General notes

    This antibody may be used to identify fibronectin during wound healing, during tumour progression, and tumour invasion respectively.

Properties

Applications

Our Abpromise guarantee covers the use of ab23750 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
ELISA 1/20000.
RIA 1/10000.
ICC/IF 1/40.
IHC-Fr 1/1000.
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function

    Fibronectins bind cell surfaces and various compounds including collagen, fibrin, heparin, DNA, and actin. Fibronectins are involved in cell adhesion, cell motility, opsonization, wound healing, and maintenance of cell shape. Involved in osteoblast compaction through the fibronectin fibrillogenesis cell-mediated matrix assembly process, essential for osteoblast mineralization. Participates in the regulation of type I collagen deposition by osteoblasts.
    Anastellin binds fibronectin and induces fibril formation. This fibronectin polymer, named superfibronectin, exhibits enhanced adhesive properties. Both anastellin and superfibronectin inhibit tumor growth, angiogenesis and metastasis. Anastellin activates p38 MAPK and inhibits lysophospholipid signaling.
  • Tissue specificity

    Plasma FN (soluble dimeric form) is secreted by hepatocytes. Cellular FN (dimeric or cross-linked multimeric forms), made by fibroblasts, epithelial and other cell types, is deposited as fibrils in the extracellular matrix. Ugl-Y1, Ugl-Y2 and Ugl-Y3 are found in urine.
  • Involvement in disease

    Glomerulopathy with fibronectin deposits 2
  • Sequence similarities

    Contains 12 fibronectin type-I domains.
    Contains 2 fibronectin type-II domains.
    Contains 16 fibronectin type-III domains.
  • Developmental stage

    Ugl-Y1, Ugl-Y2 and Ugl-Y3 are present in the urine from 0 to 17 years of age.
  • Post-translational
    modifications

    Sulfated.
    It is not known whether both or only one of Thr-2064 and Thr-2065 are/is glycosylated.
    Forms covalent cross-links mediated by a transglutaminase, such as F13A or TGM2, between a glutamine and the epsilon-amino group of a lysine residue, forming homopolymers and heteropolymers (e.g. fibrinogen-fibronectin, collagen-fibronectin heteropolymers).
    Phosphorylated by FAM20C in the extracellular medium.
    Proteolytic processing produces the C-terminal NC1 peptide, anastellin.
  • Cellular localization

    Secreted, extracellular space, extracellular matrix.
  • Information by UniProt
  • Database links

  • Alternative names

    • CIG antibody
    • Cold insoluble globulin antibody
    • Cold-insoluble globulin antibody
    • DKFZp686F10164 antibody
    • DKFZp686H0342 antibody
    • DKFZp686I1370 antibody
    • DKFZp686O13149 antibody
    • ED B antibody
    • Fibronectin 1 antibody
    • FINC antibody
    • FINC_HUMAN antibody
    • FN antibody
    • FN1 antibody
    • FNZ antibody
    • GFND antibody
    • GFND2 antibody
    • LETS antibody
    • Migration stimulating factor antibody
    • MSF antibody
    • Ugl-Y3 antibody
    see all

Images

  • Immunofluorescence assay was performed on paired NFs (normal fibroblasts; left) and CAFs (cancer-associated fibroblasts; right) from sample ID 1 using anti-fibronectin (red).

    We isolated CAFs from 15 human lung carcinomas and their corresponding counterpart NFs from their matched non-malignant adjacent tissues, taken at least 10 cm from the outer tumor margin. NFs and CAFs were maintained in 1:1 mixture of DMEM and F12 medium supplemented with 400 ng/ml hydrocortisone, 200 ng/ml insulin, 15% FBS and 1% penicilin/streptomycin. Cells were maintained in 5% CO2 incubator at 37°C.

    For immunofluorescence assay, samples were stained with fibronectin after blocking with bovine serum albumin. Samples were incubated with Alexa Fluor 568-conjugated secondary antibody (red). Microscopic observation was performed under a fluorescence microscope.

  • ab23750 staining Fibronectin in Rat skin tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with Immunoblock® (5 µg/ml) for 30 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/500 in diluent) for 1 hour 30 minutes at 25°C. An undiluted Goat anti-rabbit HRP/DAB polydetector polyclonal was used as the secondary antibody.

    See Abreview

  • ab23750 staining fibronectin in mouse liver tissue by immunohistochemistry (frozen sections). Cells were formaldehyde fixed and permeabilized in 0.2% Triton X-100 prior to blocking in 2% BSA for 30 minutes at 20°C. The primary antibody was diluted 1/200 and incubated with the sample for 9 hours at 4°C. Alexa fluor® 488 goat polyclonal to rabbit Ig, diluted 1/200, was used as the secondary antibody.

    See Abreview

  • ab23750 staining Fibronectin in mouse bone marrow cells by Immunocytochemistry/ Immunofluorescence. Cells were formaldehyde fixed, blocked in 2% BSA for 45 minutes at 20°C. The primary antibody was diluted 1/200 and incubated with sample for 9 hour at 4°C. An Alexa Fluor® 488 conjugated goat polyclonal to rabbit IgG, was used undiluted as secondary. The nuclei were stained with DAPI.

  • ab23750 (2ug/ml) staining Fibronectin in human aorta using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining Fibronectin in the cytoplasm and extracellularly in the connective tissue.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

References

This product has been referenced in:

See all 111 Publications for this product

Customer reviews and Q&As

1-10 of 13 Q&A

Answer

Thank you for contacting us.

This antibody has been characterised in Western Blot using 5% BSA as a blocking agent (https://www.abcam.com/Fibronectin-antibody-ab23750.html#Fibronectin-Primary-antibodies-ab23750-2.jpg). It has also been reported that ab23750 also works in Western Blot using 5% milk (see end-user feedback https://www.abcam.com/index.html?datasheet=23750&tab=abreviews&intabreviewid=18836).

I would recommend to try 5% BSA if milk is not providing satisfactory results. The use of BSA may reduce the background.
I would also suggest to check that the tranfer of the Fibronectin from the gel to the membrane is ok.

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

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Answer

r/>
I can confirm the Safety Data Sheets can be found on the online datasheets. Near the top right hand side of the datasheet, there are two 'buttons', one for Datasheet PDF and the one underneath says SDS. Click on the SDS button to link to the document.

I hope this will be helpful. If you have any difficulties finding these, or if you have any further questions, please do not hesitate to contact me.

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Answer

Thank you for contacting us.

I do understand and appreciate the amount of time you have spent it the laboratory. I am sorry to hear you are disappointed with our service.


If a particular antibody causes problems we do encourage customers to contact us as soon as possible to save time and material.

To issue a replacement or a refund, we have to make sure we have ruled out possible protocol problems. We also depend on the customer to give us the information we need to speed up this process. Mr R.s protocol looks fine, therefore, there is no need for protocol tips which would be a waste of more material and time.


Regarding the quality of our products, I would like to point out that we have even extended our Abpromise period from 4 months up to 6 months now, as we are feeling confident in our products. Further on, I ran a complaint counter for the ab23750 Anti-Fibronectin antibody from July 2005 (published in catalogue) to this date, and this complaint is the thirdcomplaint for these antibody so far over all this time. In addition, we have sold over 40 vials of this antibody in the last 3 month alone. Therefore, it is very regrettable that the last product received don’t seem to work and we are keen on finding the reason why. We will use thosedata internally to investigate the source of the problem with this product as well.

If these investigation point out that a product is constantly missing our quality target,and fails to improve, we usually initiate a product recall and inform our customers and offera refund, credit note orreplacement. We will not, however, compensate for more than the ordered antibody, as we state this in our Terms and Conditions.

I found an order for the EMBL in Heidelberg which was issued on the XXXXX. I this the particular order? If this is the case, I will issue the refund immediately.

I thank you for your understanding and cooperation. I look forward to hearing from you.

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Answer

Thank you for taking time to answer my questions.


Having reviewed the additional protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement, credit note, or refund in compensation (when the product has been purchased in the last 180 days). I would need the original order number or the date of the order to arrange this

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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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab23750 Anti-Fibronectin antibody. I would also appreciate if you can confirm some further details:

1. I would like to know a bit more about the samples you have used.

What kind of species and what kind of sample are you using? Fibronectincomes in many Isoforms, of which a a few are sample depended. In addition, the protein is heavily n- and o-glycosylated which cancause a ladder effect.
What was the lysis buffer? Did you use protease inhibitors?
Are you boiling your samples? If so, what's the reducing buffer, for how long and at which temperature?
How much protein was loaded?



2.Having never worked with the iBLOT system myself I am wondering how reliable it is in terms of transferring heavy proteins?They can be a bit tricky in normal semi-dry blotting as well compared to proteins like Actin.Have you checked thetransfer?

3. What are the gels running conditions?

4. Have you performed a non-primary control?


In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for your enquiry.

Although we produce many antibodies in house, I am sorry to confirm that this particular antibody has been tested externally, and regrettably we have no images of western blot on mouse samples.

However, I would like to reassure you that our antibodies have all been tested successfully and would be covered by our 6 month guarantee in the applications and species listed on the datasheets. Therefore, ab23750 Fibronectin antibody will be covered by the guarantee in western blot and on mouse samples.

For further information regarding the guarantee:

https://www.abcam.com/index.html?pageconfig=abpromise

If you would like to try the antibody in western blot and mouse, you may like to consider providing some feedback and an image. We would be very grateful if you could submit an Abreview about this product via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers.

To find out more about our Abreview system including Abpoints rewards for submitting feedback, please see the following webpage:

https://www.abcam.com/abreviews

I hope this will be helpful to you and that our guarantee will provide some reassurance. Should you have any further questions please do not hesitate to ask.

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Answer

Indeed, I can confirm that the immunogen shares a sequence similarity of 97% with rat and it is therefore likely that the antibody cross-reacts. DISCOUNT CODE: xxx Expiration date: dd mm 2012 I am very pleased to hear you would like to accept our offer and test ab23750 in rat. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for rat and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount  

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Answer

I recently "published" your Abreview on ab23750 on our website. Thank you for sharing your data with us! I just wanted to follow up with as you had rated your experience with this antibody as 'below average'. Since the staining was specific but lacked intensity, signal may be increased by decreasing the dilution to 1:50 and to incubate the primary overnight at 4 degrees C with your sample. I look forward to hearing if this advice proves helpful.

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Answer

Thank you for contacting us. Abcam takes product quality seriously and we recently undertook an in-depth study examining storage conditions and antibody performance. After over 13,000 individual ELISA experiments, we have determined that storing our antibodies at various temperatures of up to 45°C for 1 week does not impact their activity. I would be happy to share a summary of our findings with you; please let me know if you would like to see this. As a precautionary measure only, we ship our antibodies in packaging with ice packs to provide extra temperature stability in transit. The ice pack may be thawed or even at room temperature when you receive it; please be assured that this is normal and that your product is safe to use. Once you have received the vial, please follow the long-term storage instructions on the datasheet. Our Abpromise to you is that, should our product not work as stated on the datasheet, we will resolve the issue to your satisfaction, either with helpful advice to optimize your experiment or a replacement or refund of the product. Please contact our knowledgeable Scientific Support staff should you experience any issues while using our products. Contact information can be found at: https://www.abcam.com/index.html?pageconfig=contactus I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. PS: Attached is the tested data of few of our antibodies.

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Answer

Thank you for contacting us. We have reports of several methods being useful for antigen retrieval. The two main approaches are either an enzymatic retrieval using hyaluronidase, or a heat-mediated retrieval with either sodium citrate buffer at pH 6.0, or EDTA buffer at pH 9.0. The enzymatic retrieval with hyaluronidase is recommended by our source for this antibody, and is, in brief, as follows: "After de-waxing the tissue slices they are treated with 0.2% hyaluronidase (approximately 300 U/mg) in TBS 15 min at 37°C. There after non-specific binding is blocked by blocking serum or 3% BSA in TBS. For peroxidase systems, blocking with 1% peroxide solution in TBS for 30 min at RT is recommended." A customer also reports, in a review from 2006: "Hyaluronidase digestion is really necessary: 0.2% hyaluronidase (type IV-S from Bovine testes) in PBS, 15 min at 37C." The review can be found here: https://www.abcam.com/index.html?pageconfig=reviews&intAbID=23750&strFilterApplication=Immunohistochemistry%20(Formalin/PFA-fixed%20paraffin-embedded%20sections) A more recent review (on the same page) from 2010 found that heat-mediated retrieval in citrate buffer was sufficient, so if this is what you are accustomed to, you may want to try this first before investing in hyaluronidase. 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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1-10 of 13 Q&A

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