ATL4 Antibody

What is ATL4 antibody and what protein does it target?

ATL4 antibody is a research-grade immunological reagent designed to detect and bind to the ADAMTSL4 protein (also known as TSRC1, PP1396, or UNQ2803/PRO34012). The antibody specifically targets synthetic peptides derived from human ATL4 and shows reactivity across human, mouse, and rat samples . ADAMTSL4 belongs to the ADAMTS-like protein family, which has been implicated in extracellular matrix organization and various developmental processes.

What applications is the ATL4 antibody validated for?

Based on available product information, ATL4 antibody (catalog number C40337) is primarily validated for Western Blot (WB) applications at dilutions of 1:500-2000 . For researchers considering other applications such as immunohistochemistry, immunofluorescence, or ELISA, it is essential to conduct validation studies before proceeding with experiments, as application-specific validation is a critical determinant of antibody performance and reproducibility in scientific research .

What are the key technical specifications of commercially available ATL4 antibodies?

The ATL4 antibody described in the literature is a polyclonal antibody raised in rabbits and affinity-purified using a specific immunogen (synthesized peptide derived from human ATL4). It is typically supplied at a concentration of 1 mg/ml in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide. The antibody detects endogenous levels of ATL4 in human, mouse, and rat samples and has a recommended storage temperature of -20°C with a shelf life of approximately one year .

How should researchers validate ATL4 antibody specificity before experimental use?

After selecting an ATL4 antibody, researchers should perform context-specific validation for their particular research question and experimental system . A comprehensive validation approach includes:

• Positive and negative controls: Use samples with known expression levels of ADAMTSL4, including knockout or knockdown models where available

• Blocking peptide competition: Competition with the immunizing peptide should abolish specific binding

• Multiple detection methods: Confirm results using orthogonal techniques (e.g., mass spectrometry)

• Reproducibility testing: Ensure consistent results across multiple experimental replicates

• Molecular weight verification: Confirm that the detected band matches the expected molecular weight of ADAMTSL4

This thorough validation process helps ensure that experimental results accurately reflect ADAMTSL4 biology rather than non-specific binding or artifacts.

What criteria should guide the selection of an appropriate ATL4 antibody for specific research applications?

When selecting an ATL4 antibody, researchers should evaluate several critical factors:

• Validation data quality: Review all available validation images and supporting data provided by the manufacturer

• Application suitability: Ensure the antibody has been validated for your specific application (e.g., Western blot, IHC, IF)

• Species reactivity: Verify cross-reactivity with your experimental model organism

• Clonality consideration: Determine whether polyclonal or monoclonal antibodies are more appropriate for your research question

• Manufacturer reputation: Purchase from transparent companies that disclose antigen information and provide comprehensive validation data

• Literature precedent: Review published studies that have successfully used the antibody

Finding an antibody that meets these criteria increases the likelihood of obtaining reproducible and reliable results in ATL4 research.

What are the optimal conditions for Western blot analysis using ATL4 antibody?

For optimal Western blot results with ATL4 antibody, researchers should consider the following protocol modifications:

Sample Preparation and Electrophoresis:

• Use fresh tissue/cell lysates prepared with protease inhibitors

• Determine optimal protein loading (typically 20-50 μg of total protein)

• Select appropriate gel percentage based on ADAMTSL4's molecular weight

Blotting and Detection Parameters:

• Transfer time: 60-90 minutes at 100V or overnight at 30V (4°C)

• Blocking: 5% non-fat dry milk or BSA in TBST (1 hour, room temperature)

• Primary antibody: ATL4 antibody at 1:500-1:2000 dilution

• Incubation time: Overnight at 4°C

• Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000

• Detection: Enhanced chemiluminescence (ECL) substrate

The optimal dilution should be determined empirically for each new lot of antibody to ensure consistent and specific detection of ADAMTSL4.

How can immunoprecipitation experiments with ATL4 antibody be optimized?

While the specific ATL4 antibody mentioned (C40337) is not explicitly validated for immunoprecipitation, researchers attempting this application should consider these optimization strategies:

• Pre-clearing: Remove non-specific binding proteins from lysates before adding ATL4 antibody

• Antibody amount: Titrate antibody amount (typically 1-5 μg per 100-500 μg of total protein)

• Incubation conditions: Test both short (2-4 hours) and long (overnight) incubations at 4°C

• Bead selection: Compare protein A, protein G, or combination beads for optimal pull-down

• Wash stringency: Balance between reducing background and maintaining specific interactions

• Elution method: Test both gentle (non-denaturing) and harsh (denaturing) elution conditions

Researchers should validate immunoprecipitation efficiency through Western blot analysis of input, unbound, and eluted fractions to confirm specific enrichment of ADAMTSL4.

How should researchers analyze and interpret Western blot data generated with ATL4 antibody?

Proper analysis of Western blot data for ADAMTSL4 requires:

• Molecular weight verification: ADAMTSL4 has predicted molecular weights that should be confirmed against detected bands

• Quantification approach: Use appropriate software (ImageJ, Image Lab, etc.) for densitometric analysis

• Normalization strategy: Normalize ADAMTSL4 signals to appropriate loading controls (β-actin, GAPDH, etc.)

• Statistical analysis: Apply appropriate statistical tests based on experimental design, such as Friedman's test for comparing multiple techniques or Wilcoxon's matched-pairs signed-rank test for pairwise comparisons

• Biological replicates: Include sufficient replicates (minimum n=3) for robust statistical analysis

When analyzing ADAMTSL4 expression patterns, researchers should consider potential post-translational modifications that might affect apparent molecular weight and antibody recognition.

What statistical approaches are most appropriate for analyzing antibody-based experimental data?

The statistical approach should be tailored to the experimental design and data characteristics:

For parametric alternatives, researchers should first verify that data meet normality assumptions using appropriate tests (Shapiro-Wilk, Kolmogorov-Smirnov). Transformations (e.g., log transformation) may help achieve normality for statistical analysis when appropriate .

What are common issues encountered with ATL4 antibody and how can they be resolved?

Researchers working with ATL4 antibody may encounter several common challenges:

• Weak or no signal:

  • Increase antibody concentration or extend incubation time

  • Verify protein transfer efficiency with reversible staining

  • Confirm sample preparation preserves ADAMTSL4 protein integrity

  • Consider using enhanced detection systems (e.g., amplified chemiluminescence)

• Multiple bands or high background:

  • Increase blocking time or blocking agent concentration

  • Optimize antibody dilution (typically 1:500-2000 for Western blot)

  • Use more stringent washing conditions

  • Filter antibody solution to remove aggregates

• Inconsistent results between experiments:

  • Standardize lysate preparation protocols

  • Use positive controls in each experiment

  • Maintain consistent exposure times during imaging

  • Implement quality control checkpoints throughout the protocol

Systematic troubleshooting approaches that modify one variable at a time help identify the source of experimental issues.

How can researchers ensure long-term stability and reproducibility when working with ATL4 antibody?

To maintain ATL4 antibody performance over time:

• Storage conditions: Store antibody at -20°C as recommended, avoiding repeated freeze-thaw cycles by preparing small working aliquots

• Handling practices: Maintain cold chain during experiments, avoid contamination, and use sterile technique

• Quality control: Include positive and negative controls in each experiment to verify antibody performance

• Lot testing: Validate each new lot against previous lots before use in critical experiments

• Documentation: Maintain detailed records of antibody source, lot number, and performance in each application

• Standardization: Use consistent protocols, reagents, and equipment across experiments

Implementing these practices reduces variability and increases confidence in experimental results across different time points and between researchers.

How can ATL4 antibody be used in multiplex immunoassays?

For researchers investigating ADAMTSL4 in complex biological contexts, multiplex approaches offer significant advantages:

• Co-immunoprecipitation: Use ATL4 antibody to identify protein interaction partners of ADAMTSL4

• Proximity ligation assay (PLA): Combine ATL4 antibody with antibodies against suspected interaction partners to visualize protein-protein interactions in situ

• Multiplex immunofluorescence: Pair ATL4 antibody with antibodies against tissue markers to map ADAMTSL4 expression in heterogeneous tissues

• ChIP-seq applications: If ADAMTSL4 has nuclear functions, ATL4 antibody could be adapted for chromatin immunoprecipitation studies

When developing multiplex assays, researchers must first validate antibody compatibility, including:

• Absence of cross-reactivity between antibodies

• Compatibility of antibody host species

• Similar optimal fixation and antigen retrieval conditions

• Spectral separation of detection systems

What considerations are important when using ATL4 antibody for tissue microarray analysis?

When applying ATL4 antibody to tissue microarrays (TMAs), researchers should address:

• Fixation effects: Optimize antigen retrieval for formalin-fixed paraffin-embedded tissues

• Antibody titration: Determine optimal concentration specifically for TMA applications

• Scoring system: Develop and validate quantitative scoring methods for ADAMTSL4 expression

• Controls: Include positive and negative control tissues in each TMA

• Correlation analysis: Link ADAMTSL4 expression to clinical parameters and outcomes

• Heterogeneity assessment: Evaluate multiple cores per case to account for tissue heterogeneity