ARI13 Antibody

What is ARIH2/TRIAD1 and what cellular functions does it perform?

ARIH2/TRIAD1 (also known as ARI-2, Protein ariadne-2 homolog) functions as an E3 ubiquitin-protein ligase, catalyzing ubiquitination of target proteins in conjunction with the ubiquitin-conjugating enzyme E2 UBE2L3 . It acts as an atypical E3 ubiquitin-protein ligase by working in coordination with the cullin-5-RING ubiquitin ligase complex (ECS/CRL5 complex), specifically mediating the addition of the first ubiquitin on ECS targets . Its ligase activity is activated upon binding to the neddylated form of the cullin-5 (CUL5) component of the ECS complex . Additionally, ARIH2 may play a significant role in myelopoiesis and has been implicated in viral defense mechanisms during HIV-1 infection .

What are the common applications for ARIH2/TRIAD1 antibodies?

ARIH2/TRIAD1 antibodies are primarily used in Western blotting (WB) applications with human and rat samples . The common recombinant monoclonal antibody (such as EPR7671) has been validated for detecting ARIH2/TRIAD1 in cell lysates from various human cell lines including 293T, Jurkat, HeLa, and rat PC12 cells . The predicted band size for ARIH2/TRIAD1 is approximately 57 kDa, which helps researchers confirm the specificity of antibody binding in Western blot applications . These antibodies are valuable tools for investigating the role of ARIH2 in ubiquitination pathways and its interactions with various cellular components.

How does ARIH2/TRIAD1 interact with other proteins in ubiquitination pathways?

ARIH2/TRIAD1 works cooperatively with the ECS complex to initiate ubiquitination of target substrates . After ARIH2 adds the initial ubiquitin, this modification is then elongated by other components of the ubiquitination machinery . ARIH2 specifically catalyzes 'Lys-6', 'Lys-48'- and 'Lys-63'-linked polyubiquitination, suggesting diverse roles in protein degradation and signaling pathways . In conjunction with the ECS(ASB9) complex, it catalyzes ubiquitination of CKB and also promotes ubiquitination of DCUN1D1 . During HIV-1 infection, ARIH2 works with a cullin-5-RING E3 ubiquitin-protein ligase complex hijacked by the HIV-1 Vif protein to catalyze ubiquitination and degradation of APOBEC3F and APOBEC3G, highlighting its role in viral defense mechanisms .

What methodologies are most effective for validating antibody specificity for ARIH2/TRIAD1?

The most robust approach to antibody validation involves using multiple complementary methods in conjunction with appropriate positive and negative controls. For ARIH2/TRIAD1 antibodies, effective validation should include:

• Western blotting with verified positive controls (cells expressing ARIH2) and negative controls (cells with confirmed absence of ARIH2)

• Immunohistochemistry (IHC) on control tissues with known expression patterns

• Immunoprecipitation followed by mass spectrometry (IP-MS) to confirm antibody-target interaction

• Correlation of protein detection with mRNA expression levels

This multi-method approach can help identify issues with antibody specificity, as demonstrated in studies of oestrogen receptor beta antibodies where some commercially available antibodies failed comprehensive validation tests despite being widely used in research . For ARIH2 antibodies, validation should verify the detection of the expected 57 kDa band in Western blots and absence of cross-reactivity with other proteins .

How can researchers address discrepancies between antibody-based protein detection and mRNA expression data?

Discrepancies between protein detection via antibody-based methods and mRNA expression represent a significant challenge in research. To address this issue:

• Validate antibody specificity using both positive and negative controls with confirmed expression status

• Employ multiple antibodies targeting different epitopes of the same protein

• Conduct IP-MS to confirm the identity of the protein being detected

• Use complementary methods such as RNA interference or CRISPR-based gene editing to modulate protein expression and confirm antibody specificity

A study on oestrogen receptor beta found that only one of 13 tested antibodies demonstrated congruence between IHC positivity and mRNA expression levels . The high-quality antibody (PPZ0506) showed positive staining only in tissues with detectable transcript levels, while other commonly used antibodies resulted in positive staining in tissues lacking detectable mRNA . This highlights the importance of correlating protein detection with independent measures of gene expression.

What factors contribute to antibody instability and loss of specificity over time?

Antibody stability and specificity can deteriorate over time due to several factors:

• Storage conditions - improper temperature, freeze-thaw cycles

• Buffer composition - pH changes, preservative degradation

• Protein aggregation and denaturation

• Contamination with microorganisms or proteases

Research has documented that even well-characterized antibodies can lose specificity after prolonged storage. For example, the 14C8 antibody for ERβ was found to lose its ability to recognize recombinant ERβ after months of storage, rendering it unable to distinguish between positive and negative controls . To minimize these issues, researchers should:

• Aliquot antibodies to minimize freeze-thaw cycles

• Store at recommended temperatures (typically -20°C or -80°C for long-term storage)

• Validate older antibody stocks against fresh controls before use in critical experiments

• Document lot numbers and maintain validation data for each antibody batch

What are the optimal conditions for Western blot analysis using ARIH2/TRIAD1 antibodies?

For optimal Western blot results with ARIH2/TRIAD1 antibodies, researchers should consider the following protocol elements:

• Sample preparation: Use cell lysates from appropriate cell lines (293T, Jurkat, HeLa, or PC12) at approximately 10 μg protein loading per lane

• Antibody dilution: For the EPR7671 antibody, a 1/1000 dilution has been validated as effective

• Secondary antibody: Use Goat anti-Rabbit HRP at 1/2000 dilution

• Expected results: Look for a band at approximately 57 kDa (the predicted molecular weight of ARIH2/TRIAD1)

• Controls: Include positive control lysates from cells known to express ARIH2/TRIAD1 and negative controls when possible

Optimizing these conditions will help ensure specific detection of ARIH2/TRIAD1 and minimize background or non-specific binding that could complicate data interpretation.

How can immunoprecipitation followed by mass spectrometry (IP-MS) verify antibody specificity?

IP-MS represents a gold standard for confirming antibody-target binding and should be conducted as follows:

• Perform immunoprecipitation using the antibody of interest with lysates from positive control cells expressing the target protein

• Separate the immunoprecipitated proteins by gel electrophoresis

• Excise gel sections corresponding to the expected molecular weight range of the target protein

• Analyze the excised sections by mass spectrometry to identify bound proteins

This methodology provides direct evidence of which proteins the antibody is binding. In studies on ERβ antibodies, IP-MS confirmed that only one antibody (PPZ0506) consistently and robustly bound to ERβ protein, while another antibody (14C8) showed less reproducible binding, and a third popular antibody (PPG5/10) failed to demonstrate binding to ERβ at all . These findings correlated well with the antibodies' performance in other validation assays, highlighting the value of IP-MS as a validation tool.

What strategies can minimize batch-to-batch variability in antibody performance?

Batch-to-batch variability presents a significant challenge in antibody-based research. To address this issue, researchers should:

• Use recombinant monoclonal antibodies when possible, as they typically show higher consistency than polyclonal antibodies

• Maintain detailed records of antibody lot numbers and corresponding validation data

• Perform validation tests on each new antibody lot before use in critical experiments

• Consider developing in-house reference standards for comparing antibody performance across batches

• Use multiple antibodies targeting different epitopes of the same protein when feasible

Recombinant antibodies, such as the ARIH2/TRIAD1 antibody [EPR7671], offer advantages in consistency and affinity . Research has shown that recombinant antibodies appear to have, on average, 1-2 orders of magnitude higher affinity compared to traditional mouse monoclonal antibodies . This higher affinity and batch-to-batch consistency make recombinant antibodies particularly valuable for quantitative applications and longitudinal studies.

How is artificial intelligence being applied to antibody design and validation?

Artificial intelligence (AI) is revolutionizing antibody research through several approaches:

• De novo generation of antigen-specific antibody sequences using germline-based templates

• AI-driven prediction of antibody-antigen interactions and binding affinities

• Computational optimization of antibody properties such as stability and specificity

Recent research has demonstrated AI-based technologies for generating antigen-specific antibody CDRH3 sequences that can bind to specific targets like SARS-CoV-2 . These AI-based processes mimic the outcome of natural antibody generation while bypassing the complexity of B cell processes like germline gene recombination and somatic hypermutation . Such approaches represent efficient and effective alternatives to traditional experimental approaches for antibody discovery, potentially accelerating the development of new research and therapeutic antibodies .

What are the advantages of broadly neutralizing antibodies and how are they identified?

Broadly neutralizing antibodies (bNAbs) recognize conserved epitopes across multiple variants of a pathogen, offering protection against diverse strains. The advantages and identification methods include:

• Advantages:

  • Protection against current and future variants of pathogens

  • Potential for prophylactic and therapeutic applications

  • Reduced likelihood of escape mutations

• Identification methods:

  • Isolation from plasma of patients with hybrid immunity (infection plus vaccination)

  • Screening against panels of variant antigens to identify broad reactivity

  • Advanced technologies like Ig-Seq for precise molecular sequencing

A recent example is the SC27 antibody, discovered by researchers at the University of Texas at Austin, which can neutralize all known variants of SARS-CoV-2 as well as distantly related SARS-like coronaviruses . This antibody was isolated from a single patient as part of a study on hybrid immunity and works by binding to highly conserved regions of the spike protein that are crucial for viral entry into cells . The molecular sequence of SC27 has been determined, opening possibilities for manufacturing it on a larger scale for future treatments .

How do trispecific antibodies differ from traditional monoclonal antibodies in research applications?

Trispecific antibodies represent an advanced antibody engineering approach with distinct advantages:

• Structure and binding properties:

  • Capable of binding three different epitopes or antigens simultaneously

  • Engineered fusion proteins combining binding domains from different antibodies

  • More complex structure than traditional monoclonal or even bispecific antibodies

• Research applications:

  • Study of complex protein-protein interaction networks

  • Investigation of multi-component cellular pathways

  • Development of novel therapeutic approaches targeting multiple disease mechanisms

• Advantages over traditional antibodies:

  • Increased specificity through multiple targeting requirements

  • Potential to modulate complex cellular pathways more precisely

  • Ability to bridge multiple cellular components in proximity-based applications

How can researchers distinguish between genuine low-level protein expression and non-specific antibody binding?

Distinguishing between low-level expression and non-specific binding requires a multi-faceted approach:

• Validation with multiple antibodies targeting different epitopes of the same protein

• Correlation with mRNA expression data from the same tissues or cells

• Use of genetic manipulation (siRNA, CRISPR) to modulate protein expression

• Implementation of proper negative controls (tissues/cells known not to express the target)

• Titration of antibody concentrations to determine optimal signal-to-noise ratio

Research on ERβ antibodies demonstrated that only one antibody (PPZ0506) showed IHC positivity that correlated well with tissue RNA expression patterns . In contrast, other antibodies (14C8 and PPG5/10) showed positive staining in tissues lacking detectable ERβ transcript levels, suggesting non-specific binding rather than detection of low-level expression . This underscores the importance of correlating protein detection with independent measures of gene expression when validating antibody specificity.

What strategies help resolve contradictory results obtained with different antibodies against the same target?

When different antibodies against the same target yield contradictory results, researchers should:

• Conduct comprehensive validation of each antibody using:

  • Western blotting with positive and negative controls

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry on tissues with known expression patterns

• Compare antibody properties:

  • Epitope location (different domains may be differentially accessible)

  • Antibody format (monoclonal vs. polyclonal, antibody class, etc.)

  • Validation history and published data

• Consider technical factors:

  • Application-specific performance (some antibodies work in WB but not IHC)

  • Protein conformation in different applications

  • Post-translational modifications that may affect epitope recognition

The study on ERβ antibodies provides a clear example of this challenge, where three commonly used antibodies generated discordant results . Only through rigorous multi-method validation, including IP-MS to confirm target binding, could researchers determine which antibody (PPZ0506) provided the most accurate representation of ERβ expression .

What are the most critical considerations for selecting antibodies for ARIH2/TRIAD1 research?

When selecting antibodies for ARIH2/TRIAD1 research, researchers should prioritize:

• Validation status: Choose antibodies validated using multiple methods including Western blot, immunohistochemistry, and ideally IP-MS

• Application compatibility: Ensure the antibody is validated for your specific application (WB, IHC, IP, etc.)

• Species reactivity: Confirm the antibody recognizes ARIH2/TRIAD1 in your species of interest

• Controls: Have appropriate positive and negative controls available to validate performance in your experimental system

• Batch consistency: Consider using recombinant monoclonal antibodies for better batch-to-batch consistency