Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Fibronectin antibody [EPR23110-46] (ab268020)

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Overview

  • Product name

    Anti-Fibronectin antibody [EPR23110-46]
    See all Fibronectin primary antibodies

  • Description

    Rabbit monoclonal [EPR23110-46] to Fibronectin

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IHC-P, IHC-Fr, ICC/IF, WBmore details
    Unsuitable for: Flow Cyt or IP

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human Fibronectin aa 2300-2400. The exact sequence is proprietary.
    Database link: P02751

  • Positive control

    • WB: Human serum, kidney and stomach lysate. Mouse, human and rat plasma lysate. NIH/3T3 whole cell lysate; HAP1 whole cell lysate. IHC-P: Human mammary gland tissue. Human esophageal squamous cell carcinoma tissue. Mouse and rat stomach tissue. ICC/IF: HepG2 and NIH/3T3 cells.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

Our Abpromise guarantee covers the use of ab268020 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 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
IHC-Fr 1/500.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
ICC/IF 1/50.
WB 1/1000. Detects a band of approximately 285 kDa (predicted molecular weight: 262 kDa).

This antibody can be used at 0.585 μg/ml.
  • Application notes
    Is unsuitable for Flow Cyt or IP.

    • 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

      see all

    • 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

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the smooth muscle of rat stomach (PMID:650101) Counterstained with hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the smooth muscle of mouse stomach (PMID:650101) Counterstained with hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunohistochemical analysis of paraffin-embedded human esophageal squamous cell carcinoma tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the stroma of human esophageal squamous cell carcinoma (PMID:30314454). Counterstained with hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunohistochemical analysis of paraffin-embedded human mammary gland tissue labeling Fibronectin with ab268020 at 1/2000 dilution (0.29 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the stroma of human mammary gland (PMID:650101). Counterstained with hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling Fibronectin with ab268020 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HepG2 cells is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

    • Immunohistochemistry (Frozen sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunohistochemistry (Frozen sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat colon tissue labeling Fibronectin with ab268020 at 1/100 dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Positive staining mainly on smooth muscle of rat colon (PMID: 17881565) is observed. The nuclear counterstain is DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution

      Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

    • Western blot - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Western blot - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      All lanes : Anti-Fibronectin antibody [EPR23110-46] (ab268020) at 0.585 µg/ml (1:1000 dilution)

      Lane 1 : Human stomach tissue lysate at 10 µg
      Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg
      Lane 3 : Fibronectin-1 knockout HAP1 whole cell lysate at 40 µg
      Lane 4 : Wild-type HAP1 whole cell lysate at 40 µg

      Developed using the ECL technique.

      Predicted band size: 262 kDa
      Observed band size: 285 kDa
      why is the actual band size different from the predicted?



      ab268020 was shown to specifically react with Fibronectin in wild-type HAP1 cells as signal was lost in Fibronectin knockout cells. Wild-type and Fibronectin knockout samples were subjected to SDS-PAGE. ab268020 and ab129002 (Rabbit anti-Vinculin loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/5000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

      Exposure time: Lanes 1-2: 37 seconds; Lanes 3-4: 3 minutes.

      Blocking/diluting buffer and concentration: 5% NFDM/TBST.

       

    • Western blot - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Western blot - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      All lanes : Anti-Fibronectin antibody [EPR23110-46] (ab268020) at 1/1000 dilution

      Lane 1 : Human serum
      Lane 2 : Human plasma
      Lane 3 : Human kidney tissue lysate
      Lane 4 : Human stomach tissue lysate
      Lane 5 : Mouse plasma
      Lane 6 : Rat plasma

      Lysates/proteins at 20 µg per lane.

      Secondary
      Lanes 1-4 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
      Lanes 5-6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 262 kDa
      Observed band size: 258 kDa why is the actual band size different from the predicted?



      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure times: Lanes 1-2, 5-6: 3 seconds; Lane 3: 125 seconds; Lane 4: 33 seconds.

      The expression molecular weight observed is consistent with what has been described in the literature (PMID:28367057).

    • Immunohistochemistry (Frozen sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunohistochemistry (Frozen sections) - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse colon tissue labeling Fibronectin with ab268020 at 1/100 dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Positive staining mainly on smooth muscle of mouse colon (PMID: 17881565) is observed. The nuclear counterstain is DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution

      Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

    • Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [EPR23110-46] (ab268020)
      Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [EPR23110-46] (ab268020)

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling Fibronectin with ab268020 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cells is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References

    Publishing research using ab268020? Please let us know so that we can cite the reference in this datasheet.

    ab268020 has not yet been referenced specifically in any publications.

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