abitram Antibody

What is abitram protein and why is it studied?

Abitram (also known as FAM206A, CG-8, and Simiate) is a protein encoded by the ABITRAM gene. It is studied across multiple species including humans and zebrafish (Danio rerio) . While detailed functional studies are still emerging, abitram is being investigated for its biological roles in various cellular processes. The protein contains specific sequence domains that are highly conserved across species, with mouse and rat orthologs showing approximately 92% sequence identity to human abitram . Research involving abitram antibodies enables scientists to study protein localization, expression patterns, and potential functions in normal physiology and disease states.

What types of abitram antibodies are commercially available for research?

Two primary abitram antibodies appear in the research literature and commercial catalogs:

Both antibodies are purified using antigen affinity methods and are intended exclusively for research applications, not for diagnostic or therapeutic procedures . The availability of species-specific antibodies allows for comparative studies across model organisms.

How should abitram antibodies be stored and handled to maintain optimal activity?

For optimal preservation of antibody functionality, abitram antibodies should be stored according to manufacturer specifications. The Cusabio antibody (CSB-PA635847XA01DIL) should be stored at -20°C or -80°C upon receipt and repeated freeze-thaw cycles should be avoided . The Invitrogen antibody (PA5-57459) can be stored at 4°C for short-term use, but long-term storage at -20°C is recommended, also avoiding freeze-thaw cycles .

The Cusabio antibody is supplied in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative , which helps maintain stability. Proper aliquoting of antibodies upon receipt can minimize the need for repeated freezing and thawing, thereby preserving antibody functionality for longer periods.

How can specificity and cross-reactivity of abitram antibodies be rigorously validated?

Validating antibody specificity is critical for ensuring experimental reliability. For abitram antibodies, a multi-faceted validation approach is recommended:

• Positive and negative controls: Use samples with known abitram expression profiles. The PA5-57459 antibody has been validated using control HEK293T lysate (vector-only) compared to abitram/FAM206A overexpression lysate .

• Knockdown/knockout validation: Utilize RNA interference or CRISPR-Cas9 systems to reduce abitram expression and confirm corresponding reduction in antibody signal.

• Multiple detection methods: Cross-validate using different techniques such as Western blot, immunocytochemistry, and immunohistochemistry, as demonstrated with the PA5-57459 antibody .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm signal reduction in the presence of the competing antigen.

• Mass spectrometry validation: Confirm that proteins immunoprecipitated by the antibody are indeed abitram/FAM206A through peptide sequencing.

This comprehensive validation approach aligns with current best practices in antibody validation, as described in the literature on computational antibody design and structure prediction .

What are the recommended protocols for using abitram antibodies in immunofluorescence studies?

Based on the successful immunofluorescence validation of the PA5-57459 antibody in U-251 MG human cells, the following protocol framework is recommended:

• Cell preparation: Culture cells on appropriate coverslips or chamber slides to 70-80% confluence.

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes.

• Blocking: Block with 5% normal serum (matching secondary antibody host) for 1 hour.

• Primary antibody incubation: Dilute PA5-57459 antibody (optimal dilution determined empirically, typically 1:100-1:500) and incubate overnight at 4°C.

• Secondary antibody: Apply fluorophore-conjugated anti-rabbit secondary antibody for 1-2 hours at room temperature.

• Nuclear counterstaining: Use DAPI or similar nuclear stain for orientation.

• Mounting and imaging: Mount with anti-fade medium and examine using confocal or fluorescence microscopy.

The published immunofluorescence results with PA5-57459 show nuclear localization of abitram with exclusion from nucleoli , providing important information about subcellular distribution that should be confirmed in your experimental system.

How can epitope mapping be performed to understand the specific binding sites of abitram antibodies?

Epitope mapping can provide crucial information about antibody-antigen interactions and is particularly relevant for abitram antibodies. Drawing from general antibody characterization approaches , the following methods can be applied:

• Peptide array analysis: Synthesize overlapping peptides spanning the abitram sequence and identify which peptides bind to the antibody.

• Mutagenesis studies: Create point mutations or deletions in recombinant abitram and assess antibody binding to identify critical residues.

• Hydrogen-deuterium exchange mass spectrometry: This technique can identify regions of the protein that are protected from exchange when bound to the antibody.

• X-ray crystallography or cryo-EM: While resource-intensive, structural determination of the antibody-antigen complex provides the most detailed epitope information.

• Computational prediction: Utilize tools similar to "Web Antibody Modeling" (WAM) or "Prediction of Immunoglobulin Structure" (PIGS) to predict antibody-antigen binding sites .

Understanding the epitope can help predict potential cross-reactivity with related proteins and can inform experimental design when using multiple antibodies simultaneously.

What are common challenges when using abitram antibodies in Western blotting and how can they be addressed?

Western blotting with abitram antibodies may present several challenges:

• Non-specific bands: If encountering multiple bands, optimize:

  • Primary antibody concentration: Test dilutions from 1:500 to 1:5000

  • Blocking conditions: Try different blockers (5% milk, 5% BSA, commercial blockers)

  • Washing stringency: Increase wash duration or detergent concentration

• Weak or no signal: For abitram detection, consider:

  • Sample preparation: Ensure proper lysis buffers compatible with nuclear proteins

  • Protein loading: Increase amount of total protein loaded (20-50 μg)

  • Transfer conditions: Optimize for high molecular weight proteins

  • Detection system: Try higher sensitivity chemiluminescent substrates

• Inconsistent results: Control for:

  • Antibody storage conditions: Aliquot to avoid freeze-thaw

  • Sample degradation: Use fresh samples with protease inhibitors

  • Lot-to-lot variability: Document antibody lot numbers

The PA5-57459 antibody has been successfully used to detect abitram in HEK293T lysates , providing a positive control reference point for expected results.

How can abitram antibodies be optimized for immunoprecipitation of protein complexes?

When using abitram antibodies for immunoprecipitation (IP) to study protein-protein interactions:

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody coupling: Covalently couple antibody to beads using cross-linkers to avoid antibody contamination in the eluted sample.

• Buffer optimization: Test different lysis and washing buffers, considering that:

  • Higher salt (150-300 mM NaCl) reduces non-specific interactions

  • Lower detergent concentrations (0.1-0.5% NP-40 or Triton X-100) may preserve weaker interactions

  • Phosphatase inhibitors may be critical if studying phosphorylation-dependent interactions

• Elution strategies: Consider native elution with immunizing peptide versus denaturing elution.

• Controls: Always include:

  • IgG control from the same species

  • Input sample (typically 5-10% of IP material)

  • Ideally, a cell line with abitram knockdown/knockout

While specific IP protocols for abitram antibodies are not detailed in the provided search results, these principles apply based on standard immunoprecipitation practices for nuclear proteins.

How can computational approaches complement experimental validation of abitram antibodies?

Computational methods can enhance experimental validation and application of abitram antibodies in several ways:

• Epitope prediction: Algorithms can predict linear and conformational epitopes on abitram protein, helping to understand potential cross-reactivity.

• Antibody structure modeling: Tools like Rosetta Antibody can predict the structural features of the antibody variable regions that recognize abitram .

• Binding affinity prediction: Computational approaches can estimate binding affinities between antibody complementarity-determining regions (CDRs) and abitram epitopes.

• Paratope characterization: Modern approaches recognize that antibody paratopes should be described as "interconverting states in solution with varying probabilities" rather than static structures .

• Cross-reactivity assessment: Sequence similarity searches can identify proteins with regions similar to the abitram epitope that might react with the antibody.

These computational approaches, while not replacing experimental validation, can provide valuable insights and help design more targeted experiments, particularly for understanding antibody specificity challenges .

How can somatic hypermutation principles be applied to develop higher-affinity abitram antibodies?

Understanding somatic hypermutation (SHM) and affinity maturation can guide strategies for developing improved abitram antibodies:

• In vitro affinity maturation: Technologies that mimic natural SHM can be employed:

  • Phage display with error-prone PCR to introduce mutations in CDR regions

  • Yeast display systems coupled with fluorescence-activated cell sorting

  • Ribosome display with random mutagenesis

• Targeted CDR modifications: Based on computational structure prediction, specific amino acid substitutions in CDRs can be introduced to enhance binding affinity .

• Selection strategies: Design selection protocols that:

  • Progressively decrease antigen concentration to select for higher affinity

  • Include competitive elution to identify antibodies with slower off-rates

  • Incorporate negative selection against similar proteins to improve specificity

• Combinatorial approaches: Combining the best heavy and light chain variants can produce antibodies with superior binding properties.

These strategies leverage the natural principles of antibody diversification and selection to create research reagents with improved sensitivity and specificity for abitram detection .

What considerations are important when using abitram antibodies across different model organisms?

When applying abitram antibodies across species, several factors require careful consideration:

• Sequence conservation analysis: While abitram shows 92% sequence identity between human and mouse/rat orthologs , differences in specific epitopes may affect antibody binding. The zebrafish-specific antibody (CSB-PA635847XA01DIL) is designed for a more divergent ortholog .

• Validation requirements: Each antibody must be separately validated in each species:

  • Western blot to confirm correct molecular weight

  • Immunostaining to verify expected localization patterns

  • Appropriate controls specific to each species

• Application-specific optimization: Different protocols may be required for:

  • Tissue fixation methods

  • Antigen retrieval techniques

  • Blocking reagents

  • Detection systems

• Cross-species reactivity: If using a human-reactive antibody like PA5-57459 in other species, the epitope should be analyzed for conservation.

• Negative results interpretation: Lack of signal might represent genuine absence of the protein or simply lack of antibody cross-reactivity.

A systematic approach to validation across species enables comparative studies while maintaining scientific rigor.

How might custom abitram antibodies be developed for specialized research applications?

For researchers requiring antibodies with properties not available in commercial products, custom abitram antibody development might consider:

• Novel epitope selection: Target unique regions of abitram that:

  • Are accessible in the native protein conformation

  • Differ between closely related family members

  • Allow discrimination between specific post-translational modifications

• Alternative antibody formats: Beyond conventional IgG formats, consider:

  • Single-domain antibodies for improved tissue penetration

  • Bispecific antibodies for co-localization studies

  • Recombinant antibody fragments with site-specific conjugation capabilities

• Artificial antibody constructs: Leveraging recent advances in antibody engineering:

  • "Knobs-into-holes" heterodimeric formats

  • Computationally designed binding interfaces

  • Non-immunoglobulin scaffolds with antibody-like specificity

• Selection strategies: Implement phage display methods that can:

  • Identify different binding modes associated with specific ligands

  • Disentangle binding modes even for chemically similar ligands

  • Design antibodies with customized specificity profiles

These approaches could yield research tools with precisely tailored properties for specific abitram research applications.