Overview

  • Product nameAnti-Vinculin antibody [SPM227]
    See all Vinculin primary antibodies
  • Description
    Mouse monoclonal [SPM227] to Vinculin
  • Tested applicationsSuitable for: Flow Cyt, ICC/IF, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Human
    Predicted to work with: African Green Monkey
  • Immunogen

    Full length native protein (purified) corresponding to Human Vinculin.

  • Positive control
    • HeLa and 3T3 cells, Human normal prostate tissue, human lung cells; Chinese Hamster Ovary and lung

Properties

Applications

Our Abpromise guarantee covers the use of ab18058 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
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF 1/200. See Abreview.
WB Use at an assay dependent concentration. Predicted molecular weight: 130 kDa.

130kDa (vinculin) and 150kDa (meta-vinculin)

IHC-P 1/25. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • FunctionActin filament (F-actin)-binding protein involved in cell-matrix adhesion and cell-cell adhesion. Regulates cell-surface E-cadherin expression and potentiates mechanosensing by the E-cadherin complex. May also play important roles in cell morphology and locomotion.
  • Tissue specificityMetavinculin is muscle-specific.
  • Involvement in diseaseDefects in VCL are the cause of cardiomyopathy dilated type 1W (CMD1W) [MIM:611407]. Dilated cardiomyopathy is a disorder characterized by ventricular dilation and impaired systolic function, resulting in congestive heart failure and arrhythmia. Patients are at risk of premature death.
    Defects in VCL are the cause of cardiomyopathy familial hypertrophic type 15 (CMH15) [MIM:613255]. It is a hereditary heart disorder characterized by ventricular hypertrophy, which is usually asymmetric and often involves the interventricular septum. The symptoms include dyspnea, syncope, collapse, palpitations, and chest pain. They can be readily provoked by exercise. The disorder has inter- and intrafamilial variability ranging from benign to malignant forms with high risk of cardiac failure and sudden cardiac death.
  • Sequence similaritiesBelongs to the vinculin/alpha-catenin family.
  • DomainExists in at least two conformations. When in the closed, 'inactive' conformation, extensive interactions between the head and tail domains prevent detectable binding to most of its ligands. It takes on an 'active' conformation after cooperative and simultaneous binding of two different ligands. This activation involves displacement of the head-tail interactions and leads to a significant accumulation of ternary complexes. The active form then binds a number of proteins that have both signaling and structural roles that are essential for cell adhesion.
    The N-terminal globular head (Vh) comprises of subdomains D1-D4. The C-terminal tail (Vt) binds F-actin and cross-links actin filaments into bundles. An intramolecular interaction between Vh and Vt masks the F-actin-binding domain located in Vt. The binding of talin and alpha-actinin to the D1 subdomain of vinculin induces a helical bundle conversion of this subdomain, leading to the disruption of the intramolecular interaction and the exposure of the cryptic F-actin-binding domain of Vt. Vt inhibits actin filament barbed end elongation without affecting the critical concentration of actin assembly.
  • Post-translational
    modifications
    Phosphorylated; on serines, threonines and tyrosines. Phosphorylation on Tyr-1133 in activated platelets affects head-tail interactions and cell spreading but has no effect on actin binding nor on localization to focal adhesion plaques.
    Aceylated; mainly by myristic acid but also small amount of palmitic acid.
  • Cellular localizationCytoplasm > cytoskeleton. Cell junction > adherens junction. Cell membrane. Cytoplasmic face of adhesion plaques. Recruitment to cell-cell junctions occurs in a myosin II-dependent manner. Interaction with CTNNB1 is necessary for its localization to the cell-cell junctions.
  • Information by UniProt
  • Database links
  • Alternative names
    • CMD1W antibody
    • CMH15 antibody
    • Epididymis luminal protein 114 antibody
    • HEL114 antibody
    • Metavinculin antibody
    • MV antibody
    • MVCL antibody
    • OTTHUMP00000019861 antibody
    • OTTHUMP00000019862 antibody
    • VCL antibody
    • VINC antibody
    • VINC_HUMAN antibody
    • Vinculin antibody
    see all

Anti-Vinculin antibody [SPM227] images

  • ab18058 staining mouse 3T3 cells by ICC/IF.  Cells were PFA fixed and permeabilized in Triton X-100 prior to blocking in 1% BSA for 30 minutes at 25°C.  The primary antibody was diluted 1/100 and incubated with the sample for 1 hour at 25°C, ab6785, diluted 1/100, was used as the secondary antibody.

    See Abreview

  • Ab18058 staining human normal prostate. Staining is localised to the nucleus and cytoplasm.
    Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, 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 we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • Overlay histogram showing HeLa cells stained with ab18058 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab18058, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • ab18058 at 1/200 staining Chinese Hamster Ovary cells by ICC/IF. The cells were permeabilized with 100% methanol and then blocked with 1% BSA/ 4% goat serum prior to incubation with the antibody for 30 minutes. A rhodamine conjugated goat antibody was used as the secondary.

    See Abreview

  • Anti-Vinculin antibody [SPM227] (ab18058) at 1/1000 dilution + Whole cell lysate prepared from human lung cells at 40 µg

    Secondary
    HRP conjugated goat polyclonal to mouse IgG at 1/1000 dilution

    Predicted band size : 130 kDa
    Observed band size : 117 kDa (why is the actual band size different from the predicted?)
    The image is a courtsey of an anonymous abreview.

    See Abreview



  • Predicted band size : 130 kDa

    Lanes: 1, 4, 7 - Hamster Lung; 2, 5, 8 - K562; 3, 6, 9 - CHO

    Lanes: 1-3: 4oC (1 freeze/thaw);  4-6: 4oC (2 freeze/thaws); 7-9 :   4oC (4 freeze/thaws) 

  • ICC/IF image of ab18058 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab18058, 10µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 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.

References for Anti-Vinculin antibody [SPM227] (ab18058)

This product has been referenced in:
  • Chernova T  et al. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease. Cell Death Differ 23:1152-64 (2016). Read more (PubMed: 26891694) »
  • Bassi C  et al. The acetyltransferase Tip60 contributes to mammary tumorigenesis by modulating DNA repair. Cell Death Differ 23:1198-208 (2016). WB ; Mouse . Read more (PubMed: 26915295) »

See all 27 Publications for this product

Product Wall

Application Western blot
Sample Human Cell lysate - whole cell (T84 (Colon) and 16HBE(lung))
Gel Running Conditions Reduced Denaturing (6% Polyacrylamid-gel)
Loading amount 75 µg
Specification T84 (Colon) and 16HBE(lung)
Blocking step Starting Block solution from Thermo fisher as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0% · Temperature: RT°C
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Submitted Oct 13 2016

Application Western blot
Sample Rat Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions Reduced Denaturing (8%)
Loading amount 20 µg
Specification Skeletal muscle
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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Submitted Feb 09 2016

Application Western blot
Sample Human Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions Reduced Denaturing (10%)
Loading amount 15 µg
Specification Skeletal muscle
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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Submitted Feb 05 2016

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (8% Gel)
Sample Rat Tissue lysate - whole (Skeletal muscle)
Specification Skeletal muscle
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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Submitted Feb 20 2015

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (8% Gel)
Sample Mouse Tissue lysate - whole (Skeletal muscle)
Specification Skeletal muscle
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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Submitted Feb 20 2015

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (6%)
Sample Mouse Cell lysate - nuclear (Mouse Pancreatic cancer)
Specification Mouse Pancreatic cancer
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Feb 21 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (8% Gel)
Sample Mouse Tissue lysate - whole (Skeletal muscle)
Specification Skeletal muscle
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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Submitted Jan 08 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Rat Cell lysate - whole cell (rat benign mammary cell line)
Loading amount 20 µg
Specification rat benign mammary cell line
Gel Running Conditions Reduced Denaturing (12.5%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Oct 22 2012

Thank you for getting back to me. I am sorry for my late reply.

I am really not sure where these experiments may be going wrong, with so many different conditions tried I really cannot explain why no staining has been observed in every case.I...

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Thank you for getting back to me.

I am sorry to hear that the new secondary antibody has not made a difference in the results obtained. I would be happy to try to help in understanding why this might be happening.I think it would be useful if...

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