VCL Antibody

What is vinculin and what are its key biological functions?

Vinculin is an actin filament (F-actin)-binding protein involved in cell-matrix adhesion and cell-cell adhesion. It functions as one of several interacting proteins that anchor F-actin to the cell membrane . Specifically, vinculin:

• Regulates cell-surface E-cadherin expression

• Potentiates mechanosensing by the E-cadherin complex

• Plays important roles in cell morphology and locomotion

• Participates in transmembrane assembly of adhesion plaques

In the assembly process, the beta subunit of integrin binds to talin, which binds to vinculin. Vinculin then interacts with alpha-actinin and possibly with itself, creating a crucial link between the extracellular matrix and the cytoplasmic microfilament system .

What is the difference between vinculin and metavinculin?

Vinculin and metavinculin are isoforms with distinct tissue distribution and molecular weights:

Metavinculin (MV) is the muscle-specific splice variant of vinculin . Some antibodies, such as VN 3-24, can specifically recognize metavinculin, making them useful for muscle-specific research applications .

What epitopes are commonly targeted by VCL antibodies?

Commercial VCL antibodies target various epitopes across the vinculin protein:

• Abcam antibodies (ab269680, ab238075): Target epitopes within amino acids 150-350 of human VCL

• R&D Systems antibody (MAB6896): Targets the C-terminal region (Lys1020-Gln1134)

• Thermofisher antibody (7414-MSM5-P1): Targets epitopes specific to the VCL/3617 clone

• DSHB antibody (VN 3-24): Recognizes an epitope present in chicken vinculin but not in human, mouse, or rat vinculin

The choice of epitope can affect antibody performance in different applications and species reactivity.

Which applications are VCL antibodies most suitable for?

VCL antibodies demonstrate versatility across multiple experimental applications:

Most VCL antibodies perform exceptionally well in Western blot applications, with multiple publications validating their use .

What are the optimal antigen retrieval methods for VCL immunohistochemistry?

Heat-induced epitope retrieval is critical for successful vinculin detection in IHC:

Method 1: Tris-EDTA (Optimal)

• Buffer: 10mM Tris with 1mM EDTA, pH 9.0

• Temperature/Time: 95°C for 45 minutes

• Cooling: Room temperature for 20 minutes

• Validated in: Multiple human tissues

Method 2: Basic Retrieval Reagent

• Reagent: Antigen Retrieval Reagent-Basic (e.g., CTS013)

• Protocol: Follow manufacturer instructions

• Example: Used successfully for human uterus sections with MAB6896

Method 3: Citrate Buffer (Alternative)

• Buffer: Citrate buffer pH 6.0

• Note: May provide less optimal results than Tris-EDTA for some antibodies

Following retrieval, antibody incubation conditions vary: for MAB6896, optimal staining was achieved at 15 μg/mL overnight at 4°C .

How should Western blot protocols be optimized for vinculin detection?

For optimal vinculin detection by Western blot:

Sample Preparation:

• Use reducing conditions (all validated protocols specify this requirement)

• Protein separation using standard SDS-PAGE or 12-230 kDa separation systems for Simple Western

• Load 5-50 μg of whole cell lysate depending on vinculin abundance

Detection Parameters:

• Membrane: PVDF provides good results for vinculin detection

• Primary antibody concentration: 0.1-2 μg/mL depending on specific antibody

• Secondary antibody: HRP-conjugated anti-mouse or anti-rabbit IgG

• Development: ECL technique with 3-minute exposure provides clear bands

Validated Cell Lines: HeLa, HepG2, U-87, THP-1, 293T, NIH/3T3, PC-3, LNCaP, U-251, HuH-7, L02, C6, HSC-T6

The expected molecular weight is 124 kDa, but actual observation may be 117-122 kDa depending on the separation system and antibody used .

Why might I observe variable molecular weights for vinculin in Western blots?

Molecular weight variability in vinculin detection has several potential causes:

• Isoform Differences:

  • Vinculin: 124 kDa (calculated)/117-122 kDa (observed)

  • Metavinculin: ~150 kDa (muscle-specific isoform)

• Technical Variations:

  • Gel percentage affects migration (8-10% gels recommended)

  • Different separation systems show slight variations (e.g., Simple Western shows bands at ~122 kDa)

  • Buffer conditions and reducing agent concentration can impact migration

• Sample-Specific Factors:

  • Post-translational modifications may alter migration

  • Tissue-specific expression patterns (particularly in muscle tissues)

  • Species differences (though the protein is highly conserved)

When reporting results, researchers should specify both the expected and observed molecular weights, as seen in the Proteintech antibody documentation (calculated: 124 kDa; observed: 117 kDa) .

How can I validate the specificity of my VCL antibody?

A multi-faceted approach ensures proper antibody validation:

Experimental Validation Methods:

• Multiple Application Testing:

  • Verify consistent results across WB, IHC, and IF applications

  • Example: ab269680 validates in WB, Protein Array, and IHC-P

• Positive Controls:

  • Use cell lines with established vinculin expression (HeLa, HepG2)

  • Include tissue controls (human uterus shows strong staining in smooth muscle cells)

• Band Size Verification:

  • Confirm the expected molecular weight (124 kDa for vinculin)

  • Note any tissue-specific bands (150 kDa for metavinculin in muscle)

• Cross-reactivity Assessment:

  • Test antibody in multiple species if cross-reactivity is claimed

  • Note species limitations (e.g., VN 3-24 works with chicken/Xenopus but not human/mouse/rat)

• Advanced Validation:

  • Protein array screening (ab238075 was tested against >19,000 human proteins)

  • Antibody registry verification (e.g., VN 3-24 has registry ID AB_2214518)

Document all validation steps thoroughly to ensure reproducibility and reliability.

How can VCL antibodies be used to study mechanotransduction?

Vinculin plays a central role in cellular mechanosensing through focal adhesions:

Research Applications:

• Focal Adhesion Dynamics:

  • Use immunofluorescence with VCL antibodies to quantify focal adhesion size, number, and distribution

  • Track changes in response to mechanical stimuli (substrate stiffness, stretch, shear stress)

  • Example: Combine with live cell imaging to monitor real-time changes

• E-cadherin Complex Analysis:

  • Investigate mechanosensing by the E-cadherin complex

  • VCL antibodies can identify changes in vinculin recruitment to adherens junctions

  • Co-immunoprecipitation to identify binding partners under different mechanical conditions

• ECM Stiffness Research:

  • "The stiffness of the extracellular matrix (ECM) controls many cellular processes... Cells detect stiffness through adhesion structures termed focal adhesions (FAs). Vinculin, an actin-binding FA protein, plays a pivotal role in FA-mediated mechanotransduction."

  • Compare vinculin localization and phosphorylation on substrates of varying stiffness

• Experimental Design Considerations:

  • Use appropriate fixation to preserve focal adhesion structure (4% PFA is standard)

  • For immunofluorescence, use confocal microscopy to resolve individual focal adhesions

  • Consider co-staining with other focal adhesion proteins (paxillin, FAK, talin)

What is the significance of anti-vinculin antibodies in systemic sclerosis research?

Recent research reveals important connections between anti-vinculin antibodies and systemic sclerosis (SSc):

Key Research Findings:

• Clinical Associations:

  • "Anti-vinculin antibody positive patients were 9.6 times as likely to have limited cutaneous disease as those who were anti-vinculin antibody negative (95% CI 1.19, 77.23; p=0.03)"

  • "They had more than triple the risk of thyroid disease (OR 4.1, 95% CI 1.27, 13.21; p= 0.02)"

  • "Anti-vinculin antibody positive patients also had 75% lower risk of lung disease (OR 0.25, 95% CI 0.07, 0.92; p=0.04)"

• Gastrointestinal Implications:

  • "Anti-vinculin antibodies associate with slower gastric transit in SSc and may provide insight into GI complications of SSc."

  • This suggests potential mechanistic links between autoimmunity against vinculin and GI motility disorders in SSc

• Detection Methodology:

  • "A second-generation enzyme-linked immunosorbent assay (ELISA) was used to measure anti-vinculin antibodies in the sera of patients"

  • "The anti-vinculin antibody assay was considered positive when the OD was ≥ 1.68"

These findings suggest that anti-vinculin antibodies could serve as biomarkers for specific SSc subtypes and associated complications.

Can serum vinculin levels serve as biomarkers for liver diseases?

Emerging research suggests vinculin as a potential biomarker for hepatocellular carcinoma (HCC) and liver cirrhosis:

Research Evidence:

• Elevated Serum Levels:

  • "In patients with HCC and liver cirrhosis, the serum vinculin levels were significantly greater than in controls (503.8±242.2 and 728.4±1044.8 vs 77.7±36.1 respectively, p<0.001)"

• Detection Method:

  • "Vinculin levels in a total number of 100 serum samples from patients with HBV/HCV-induced liver cirrhosis and HCC, as well as healthy controls, were analyzed using an enzyme-linked immunosorbent assay (ELISA)"

• Clinical Correlations:

  • "Results showed no link between serum vinculin and the clinicopathological features of HCC"

  • This suggests vinculin may be valuable for disease detection but not for staging or characterization

• Research Implications:

  • "This might point to a potential role for vinculin in the development of HCC. More research into how this protein affects the development of HCC at the molecular level could lead to better clinical treatments and the development of new molecular therapies."

The significant elevation of serum vinculin in both HCC and cirrhosis patients suggests its potential utility as a biomarker for liver disease, though further validation studies are needed.

How do vinculin antibodies contribute to studying cell migration in cancer?

Vinculin's role in cell adhesion and migration makes it particularly relevant to cancer research:

Research Applications:

• Focal Adhesion Turnover:

  • VCL antibodies can track focal adhesion assembly/disassembly during migration

  • Immunofluorescence techniques visualize changes in focal adhesion size and distribution

  • Relevant to understanding metastatic potential of cancer cells

• EMT Transitions:

  • Vinculin participates in epithelial-mesenchymal transition processes

  • Monitor vinculin redistribution during EMT using immunofluorescence

  • Western blot quantification of vinculin in different cancer stages

• Cancer Tissue Analysis:

  • IHC staining shows vinculin distribution in cancer tissues

  • Example: "Formalin-fixed, paraffin-embedded human testicular carcinoma tissue stained for Vinculin using ab269680 at 2 μg/ml"

  • Compare vinculin expression/localization between normal and cancerous tissues

• Experimental Models:

  • Cancer cell lines successfully used with VCL antibodies include:

    • HeLa (cervical cancer)

    • HepG2 (hepatocellular carcinoma)

    • U-87 (glioblastoma)

    • PC-3 (prostate cancer)

    • LNCaP (prostate adenocarcinoma)

What methodologies are used to assess anti-vinculin antibodies in patient samples?

For clinical research involving anti-vinculin antibodies:

Validated Methodologies:

• ELISA Protocols:

  • "A second-generation enzyme-linked immunosorbent assay (ELISA) was used to measure anti-vinculin antibodies in the sera of patients using a validated second-generation assay"

  • "After undergoing epitope optimization, the antigen was mobilized onto high-binding plates and blocked with 3% bovine serum albumin in PBS to counter non-specific binding"

  • "Anti-vinculin antibody levels were evaluated after 70 minutes using optic densities (OD) obtained after measuring the absorbance at 370nm"

  • "The anti-vinculin antibody assay was considered positive when the OD was ≥ 1.68 per previously published studies"

• Serum Vinculin Quantification:

  • ELISA techniques for measuring vinculin protein (rather than anti-vinculin antibodies)

  • Used successfully in HCC/cirrhosis research with significant discrimination between patient groups and controls

• Statistical Analysis:

  • Univariate linear and logistic regression analyses to examine associations between clinical features and anti-vinculin antibody positivity

  • Calculation of odds ratios with 95% confidence intervals to quantify relationships

These methodologies enable precise quantification of both vinculin protein and anti-vinculin antibodies in patient samples, facilitating research into their potential as biomarkers.