EPHA6 Antibody, Biotin conjugated

What is the basic molecular profile of EPHA6 and its antibody?

EPHA6 (Ephrin type-A receptor 6) is a receptor tyrosine kinase with a molecular weight of approximately 116 kDa that functions in signal transduction pathways. The EPHA6 antibody discussed here (CSB-PA890673LD01HU) is a biotin-conjugated rabbit polyclonal antibody raised against recombinant Human Ephrin type-A receptor 6 protein (specifically amino acids 132-279) . This receptor belongs to the ephrin receptor subfamily and contributes to contact-dependent bidirectional signaling between adjacent cells. The biotin conjugation enables versatile detection methods, particularly when combined with streptavidin-based detection systems .

How do EPHA6 receptors function in cellular signaling?

EPHA6 receptors function as part of the Eph receptor tyrosine kinase family, which binds GPI-anchored ephrin-A ligands on adjacent cells. This binding initiates bidirectional signaling, with forward signaling occurring downstream of the receptor and reverse signaling occurring downstream of the ephrin ligand . Similar to other Eph receptors such as EphA2, EPHA6 likely requires dimerization or clustering of ligands to elicit agonistic activity in cells . The receptor-ligand interaction leads to phosphorylation events that trigger downstream signaling cascades involved in various cellular processes, including cell migration, adhesion, and tissue boundary formation.

What are the recommended storage and handling conditions for maintaining antibody activity?

For optimal preservation of EPHA6 Antibody, Biotin conjugated activity, store the antibody at -20°C or -80°C upon receipt . Critical handling considerations include:

• Avoid repeated freeze-thaw cycles that can degrade antibody function

• The antibody is provided in liquid form with a specific buffer composition: 0.03% Proclin 300 as preservative, 50% Glycerol, and 0.01M PBS at pH 7.4

• Allow the antibody to equilibrate to room temperature before opening the vial

• When diluting, use appropriate buffers that maintain pH stability and protein integrity

• Consider aliquoting the stock solution into smaller volumes to minimize freeze-thaw cycles

What are the validated applications for EPHA6 Antibody, Biotin conjugated?

Based on manufacturer specifications, EPHA6 Antibody, Biotin conjugated has been validated for ELISA applications . Although not specifically validated for other techniques, biotin-conjugated antibodies generally perform well in:

• Immunohistochemistry (IHC)

• Flow cytometry

• Immunofluorescence microscopy

• Pull-down assays (when combined with streptavidin-coated beads)

• Multiplex detection systems

Each application requires specific optimization steps, including determination of optimal antibody dilution, incubation time, and appropriate blocking reagents to minimize background signals and maximize specific detection.

How should researchers design ELISA protocols using biotin-conjugated EPHA6 antibody?

When designing ELISA protocols using biotin-conjugated EPHA6 antibody, consider the following methodological approach:

• Plate Preparation: Coat high-binding 96-well plates with target antigen (for direct ELISA) or capture antibody (for sandwich ELISA)

• Blocking: Use 2-5% BSA in PBS or 5% non-fat dry milk to reduce non-specific binding

• Primary Incubation: If using in a sandwich ELISA, apply sample containing EPHA6 protein

• Detection: Apply biotin-conjugated EPHA6 antibody at optimized dilution (typically starting at 1:1000)

• Signal Development: Add streptavidin-HRP (horseradish peroxidase) conjugate

• Visualization: Add appropriate substrate (TMB for colorimetric detection)

• Detection Controls: Include multiple controls:

  • Negative control without primary antibody

  • Negative control with isotype control antibody

  • Positive control with known EPHA6-expressing samples

This approach maximizes specificity while minimizing background signal interference.

How can researchers quantitatively assess the signal-to-noise ratio when using biotin-conjugated antibodies?

For quantitative assessment of signal-to-noise ratio when using biotin-conjugated EPHA6 antibody:

• Baseline Determination: Measure signal from negative controls (no antigen, isotype control)

• Specific Signal Measurement: Measure signal from samples containing EPHA6

• Signal-to-noise Calculation: Divide specific signal by background signal

• Optimization Matrix:

• Serial Dilution Analysis: Perform serial dilutions of the sample to determine the linear detection range

• Statistical Validation: Calculate coefficient of variation between replicates (<15% is generally acceptable)

How can EPHA6 Antibody, Biotin conjugated be utilized for studying receptor internalization and trafficking?

EPHA6 Antibody, Biotin conjugated can be effectively employed to study receptor internalization and trafficking through these methodological approaches:

• Time-course Internalization Assay:

  • Label cell surface EPHA6 with biotin-conjugated antibody at 4°C (prevents internalization)

  • Shift cells to 37°C to permit internalization

  • At various time points, remove surface-bound antibody with mild acid wash

  • Permeabilize cells and detect internalized antibody using fluorescently labeled streptavidin

  • Quantify by flow cytometry or confocal microscopy

• Co-localization Studies:

  • Label cells with biotin-conjugated EPHA6 antibody and induce internalization

  • Fix and permeabilize cells

  • Counter-stain with markers for early endosomes (EEA1), late endosomes (Rab7), lysosomes (LAMP1), or recycling endosomes (Rab11)

  • Visualize using multi-channel confocal microscopy

  • Quantify co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

Similar to studies with EphA2, receptor activation may induce receptor degradation, which can be monitored by tracking the decline in total receptor levels over time .

What strategies can be employed to study EPHA6 receptor clustering using biotin-streptavidin systems?

EPHA6 receptor clustering can be studied using biotin-streptavidin systems through these methodological approaches:

• Multimerization Strategy:

  • Combine biotinylated EPHA6 antibody with streptavidin at various ratios (1:1, 2:1, 3:1, 4:1)

  • Use these complexes to induce receptor clustering on cell surfaces

  • Compare receptor activation (phosphorylation) levels using phospho-specific antibodies

  • Quantify downstream signaling events

This approach mimics strategies employed for EphA2, where biotin-streptavidin complexes enhanced receptor activation . Specifically:

• Quantification Methods:

  • Immunoblotting for phosphorylated EPHA6

  • FRET-based assays to detect receptor proximity

  • Super-resolution microscopy (STORM or PALM) to visualize clustering patterns

• Comparative Analysis Table:

How can EPHA6 Antibody, Biotin conjugated be adapted for in vivo imaging studies?

For adapting EPHA6 Antibody, Biotin conjugated for in vivo imaging studies, researchers should consider:

• Streptavidin Conjugation Strategy:

  • Conjugate biotin-EPHA6 antibody with fluorescently labeled streptavidin (e.g., AlexaFluor dyes)

  • Optimize the antibody:streptavidin ratio (typically 4:1) for maximum signal intensity

  • Purify complexes using size exclusion chromatography

• Animal Model Selection and Preparation:

  • Develop orthotopic xenograft models expressing EPHA6 (similar to EphA2 models described in the literature)

  • Consider using GFP-expressing tumor cells for dual-labeling capability

  • Ensure appropriate controls (scrambled antibody-biotin conjugates)

• Imaging Protocol:

  • Administer purified antibody-streptavidin complex intravenously

  • Allow 24-72 hours for tumor accumulation and background clearance

  • Use appropriate imaging modality (IVIS system for whole-body imaging, confocal intravital microscopy for higher resolution)

  • Quantify tumor-to-background ratio across multiple time points

• Validation Studies:

  • Perform ex vivo tissue analysis to confirm specific binding

  • Conduct immunohistochemistry on harvested tissues

  • Correlate imaging signal with actual EPHA6 expression levels determined by Western blot or PCR

This methodology builds upon successful approaches demonstrated with EphA2-targeting biotinylated agents in orthotopic models of breast and pancreatic cancers .

What are common causes of high background when using biotin-conjugated antibodies, and how can they be mitigated?

High background is a common challenge when working with biotin-conjugated antibodies. The following table outlines major causes and mitigation strategies:

For EPHA6 Antibody, Biotin conjugated specifically:

• Begin with a dilution range of 1:500-1:2000 for ELISA applications

• Implement a blocking step using buffers containing biotin-free BSA

• If applying to tissues with high endogenous biotin (kidney, liver), use specialized biotin-blocking steps

How should researchers validate the specificity of EPHA6 Antibody, Biotin conjugated?

Comprehensive validation of EPHA6 Antibody, Biotin conjugated should include:

• Positive Control Testing:

  • Test on cell lines with known EPHA6 expression (based on RNA-seq or proteomics data)

  • Compare signal intensity with EPHA6 expression levels

• Negative Control Testing:

  • Test on EPHA6 knockout/knockdown models

  • If unavailable, use cell lines known to lack EPHA6 expression

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide (the 132-279AA region of human EPHA6)

  • Compare signal with and without peptide competition

  • Signal should be significantly reduced with peptide competition

• Cross-reactivity Assessment:

  • Test against other EphA family members (particularly EphA2, EphA4)

  • Use recombinant proteins in ELISA format for quantitative comparison

• Multiple Detection Method Validation:

  • Compare results using different detection methods (WB, IF, ELISA)

  • Patterns should be consistent across methods

• Confirmation with Alternative Antibodies:

  • Compare with non-biotinylated anti-EPHA6 antibodies

  • Results should be concordant between different antibody clones

What approaches can address signal variability in long-term studies using EPHA6 Antibody, Biotin conjugated?

To address signal variability in longitudinal studies:

• Reference Standards Implementation:

  • Include a standard curve of recombinant EPHA6 protein in each experiment

  • Express results as relative units based on standard curve

  • Maintain a large batch of reference standard aliquots for consistent comparison

• Antibody Stability Monitoring:

  • Aliquot antibody preparations to minimize freeze-thaw cycles

  • Prepare a control sample with known EPHA6 expression level

  • Test antibody activity against this control periodically

  • Document signal:background ratio over time

• Normalization Strategies:

  • For Western blots: normalize to housekeeping proteins

  • For ELISA: use internal reference samples in each plate

  • For imaging: include calibration markers

• Environmental Variable Control:

  • Maintain consistent incubation temperatures (±1°C)

  • Use the same lot of detection reagents when possible

  • Control humidity variation during incubation steps

• Statistical Approach to Variability:

  • Use technical replicates (minimum 3) for each experimental point

  • Implement appropriate statistical tests for longitudinal data (repeated measures ANOVA)

  • Establish acceptable coefficient of variation thresholds (typically <15% for technical replicates)

How does the performance of EPHA6 Antibody, Biotin conjugated compare with other Eph receptor targeting strategies?

When comparing EPHA6 Antibody, Biotin conjugated with other Eph receptor targeting approaches:

What methodological considerations should be applied when using EPHA6 Antibody, Biotin conjugated for functional studies compared to detection purposes?

For functional studies versus detection purposes:

Detection Applications:

• Optimize for signal-to-noise ratio

• Focus on antibody dilution and incubation conditions

• Primary concern is specificity and sensitivity

Functional Studies:

• Consider antibody effects on receptor activity

• Determine if antibody is neutralizing or agonistic

• Assess impact of biotin-streptavidin interaction on receptor clustering

• Evaluate potential for receptor internalization upon binding

Methodological Protocol Differences:

For functional studies, researchers should validate whether the EPHA6 Antibody, Biotin conjugated acts as an agonist or antagonist of receptor function through phosphorylation assays and downstream signaling analysis, similar to approaches used with EphA2 receptor studies .

How can researchers integrate EPHA6 Antibody, Biotin conjugated into multi-parameter analysis systems?

Integration into multi-parameter analysis systems can be achieved through:

• Multicolor Flow Cytometry:

  • Combine with differently colored fluorophores on streptavidin (e.g., streptavidin-PE, streptavidin-APC)

  • Use in conjunction with other surface markers or intracellular phospho-proteins

  • Develop sequential staining protocols to prevent steric hindrance

  • Implementation example:

    • First panel: EPHA6 (biotin-streptavidin-PE) + CD31 (FITC) + Phospho-ERK (APC)

    • Second panel: EPHA6 (biotin-streptavidin-PE) + EphA2 (APC) + EphB2 (FITC)

• Multiplexed Imaging:

  • Apply multispectral imaging techniques

  • Use streptavidin conjugated to spectrally distinct fluorophores

  • Combine with tyramide signal amplification for enhanced sensitivity

  • Example workflow:

    • Primary staining with EPHA6 Antibody, Biotin conjugated

    • Sequential detection with different streptavidin-fluorophore conjugates

    • Image acquisition using multispectral imaging systems

    • Spectral unmixing to separate overlapping signals

• Protein Array Integration:

  • Include EPHA6 Antibody, Biotin conjugated in antibody arrays

  • Develop customized arrays for Eph receptor family analysis

  • Use in reverse-phase protein arrays for patient sample analysis

Similar approaches have been successfully demonstrated with EphA2-targeting biotin-conjugated agents, particularly in combination with fluorescently-tagged streptavidin for in vivo tumor targeting in orthotopic mouse models .