vrtn Antibody

What is the biological function of VRTN and why is it significant to study?

VRTN functions as a novel DNA-binding transcription factor that localizes exclusively in the nucleus, binds to DNA on a genome-wide scale, and regulates the transcription of multiple genes containing VRTN binding motifs. It plays a critical role in embryonic development, particularly in somitogenesis and the formation of thoracic vertebrae. Studies with Vrtn-null embryos demonstrate a somitogenesis defect with failure of axial rotation and fewer somites at the thoracic stage .

The significance of VRTN lies in its essential role in determining vertebral numbers in mammals. In pigs, VRTN variants are associated with thoracic vertebral number (TVN), which has economic significance in breeding. Understanding VRTN function advances our knowledge of the mechanisms controlling segment number in mammals and contributes to developmental biology research .

Immunofluorescence studies using anti-VRTN antibodies reveal that VRTN predominantly localizes to the nucleus, consistent with its function as a transcription factor. Specifically:

• In immunofluorescent staining of human U-2 OS cells, VRTN shows positivity in the nucleus but not in nucleoli .

• Immunohistochemical staining of paraffin-embedded human small intestine tissue shows both nuclear and cytoplasmic positivity in glandular cells .

This dual localization pattern (predominantly nuclear with some cytoplasmic presence) is consistent with VRTN's role as a transcription factor that may shuttle between compartments. When designing experiments to study VRTN localization, researchers should use nuclear markers for co-localization studies and consider fixation methods that preserve nuclear architecture .

How can I validate the specificity of a VRTN antibody for my experimental system?

Validating antibody specificity is critical for reliable results. For VRTN antibodies, implement the following validation strategy:

• Knockout/knockdown controls: Generate VRTN knockdown cells using siRNA or CRISPR-Cas9 technology. Signal reduction or absence in these samples confirms specificity. Research with VRTN mutant embryos demonstrates this approach's validity .

• Epitope competition assay: Pre-incubate the antibody with excess immunizing peptide. For instance, with antibody NBP2-83757, use the peptide sequence "GGQEAEEKQEKEAGRDVTAVMAPPVGASSEDVEGGPSREGALQEGATAQG" directed towards the C-terminal region of human VRTN .

• Correlation with mRNA expression: Compare antibody signal intensity with VRTN mRNA levels across different tissues. BioGPS gene expression data supports VRTN expression in NCI-H226 cells, correlating with antibody results in western blot applications .

• Multiple antibodies approach: Use antibodies targeting different VRTN epitopes and compare detection patterns. Consistent results increase confidence in specificity.

• Positive control identification: Based on published data, use human small intestine tissue (glandular cells) or U-2 OS cells as positive controls for initial validation .

What methodological approaches can be used to study VRTN's role in developmental biology?

To investigate VRTN's role in development, several methodological approaches using antibodies can be employed:

• Temporal expression analysis: Use immunohistochemistry with VRTN antibodies on serial sections of embryos at different developmental stages. This approach was used to demonstrate VRTN's essential role during the thoracic somite stage .

• Co-localization studies: Combine VRTN antibodies with markers for Notch signaling pathway components to investigate their relationship during somitogenesis. Research has shown VRTN relates to Notch signaling molecules specifically at the thoracic somite stage .

• Conditional knockout models: Generate tissue-specific or time-controlled VRTN knockout models and use antibodies to confirm protein loss. Studies show that half of Vrtn-null embryos die, highlighting the importance of controlled experimental designs .

• ChIP-seq experiments: Use VRTN antibodies for chromatin immunoprecipitation followed by sequencing to identify genome-wide VRTN binding sites. This approach helps identify VRTN target genes involved in somitogenesis.

• Luciferase reporter assays: Similar to approaches used for studying VRTN promoter activity, antibody-based detection can be incorporated into reporter assays to correlate protein expression with functional outcomes .

How can antibody-based methods be used to investigate the relationship between VRTN variants and phenotypes?

Research has identified VRTN variants associated with phenotypic differences, particularly in thoracic vertebral number (TVN). To study these relationships using antibodies:

• Quantitative immunohistochemistry: Compare VRTN protein levels in tissues from subjects with different VRTN variants. This can be accomplished through digital image analysis of immunostained sections.

• Genotype-phenotype correlation studies: Combine genotyping of VRTN variants with quantitative antibody-based detection methods to establish correlations. Research has demonstrated that two causative variants in the regulatory region of VRTN additively regulate TVN .

• Allele-specific expression analysis: Use antibodies that can distinguish between variant forms of VRTN protein (when amino acid changes are involved) to study their differential expression.

• Functional binding assays: Study how variants affect VRTN's ability to bind target DNA sequences using antibody-based detection in electrophoretic mobility shift assays (EMSA) or ChIP experiments.

• Structure-function analysis: For variants that affect protein structure, use epitope-specific antibodies to investigate how structural changes impact VRTN's functional properties.

Research has shown that the phenotypic variance explained by VRTN variants can be estimated using the formula:
Var% = (Vreduce - Vfull)/Vreduce × 100
Where Vfull and Vreduce are the residual variances of linear models with and without VRTN variant genotypes as predictor variables .

What are the optimal conditions for using VRTN antibodies in different applications?

Optimal conditions vary by application and specific antibody:

For negative controls in immunofluorescence, replace the primary antibody with normal non-immune serum as demonstrated in developmental studies with pig embryos .

What controls should be included when designing experiments with VRTN antibodies?

Proper controls are essential for interpreting antibody-based experimental results:

• Negative controls:

  • Omission of primary antibody

  • Isotype control (irrelevant antibody of same isotype)

  • Normal non-immune serum substitution (as used in pig embryo studies)

  • VRTN-knockout or knockdown samples (when available)

• Positive controls:

  • Human small intestine tissue (shows nuclear and cytoplasmic positivity in glandular cells)

  • U-2 OS cell line (shows nuclear positivity)

  • NCI-H226 and HCT116 cells for western blot applications

• Technical controls:

  • Loading controls for western blots

  • Nuclear staining (DAPI) for localization studies

  • Serial dilution of antibody to establish optimal concentration

• Validation controls:

  • Peptide competition assay using the immunizing peptide

  • Comparison with mRNA expression data from BioGPS or other sources

How do storage conditions and handling affect VRTN antibody performance?

Based on manufacturer recommendations, optimal storage and handling of VRTN antibodies include:

• Storage temperature: Store at 4°C for short-term use. For long-term storage, aliquot and maintain at -20°C to avoid freeze-thaw cycles .

• Formulation considerations: Commercial VRTN antibodies are typically formulated in PBS with 2% sucrose and 0.09% sodium azide as a preservative .

• Aliquoting protocol: To prevent protein degradation from repeated freeze-thaw cycles, divide antibodies into single-use aliquots upon receipt and store at -20°C.

• Working dilution preparation: Always prepare working dilutions fresh on the day of experiment using appropriate diluent buffer.

• Shipping conditions: Antibodies are typically shipped with polar packs and should be stored immediately at recommended temperatures upon receipt .

Proper storage and handling significantly impact antibody performance and experiment reproducibility. Degraded antibodies may show reduced sensitivity, increased background, or complete loss of specific signal.

How can researchers interpret discrepancies in results obtained from different VRTN antibodies?

When faced with discrepancies between different VRTN antibodies, consider these factors:

• Epitope differences: Antibodies targeting different regions of VRTN may yield different results. For example, NBP2-83757 targets a C-terminal peptide , while others may target N-terminal or internal epitopes.

• Post-translational modifications: Modifications may mask certain epitopes. Compare antibodies that recognize different regions to detect potential modification-dependent differences.

• Isoform specificity: Determine if discrepancies might be due to differential recognition of VRTN isoforms.

• Fixation sensitivity: Some epitopes are more sensitive to fixation methods. Test multiple fixation protocols when results differ between applications.

• Cross-reactivity profiles: Validate specificity using multiple approaches as outlined in question 3.1.

When publishing discrepant results, document all antibody details including catalog numbers, dilutions, and application-specific conditions. Consider using antibody validation reporting standards such as those proposed in the research community.