RELA Recombinant Monoclonal Antibody

What is RELA and what is its biological significance?

RELA (V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A), also known as p65, is a critical component of the NF-κB complex that functions as a ubiquitous transcription factor. It serves as a central regulator of immune responses, inflammation, and various cellular processes including differentiation, cell growth, tumorigenesis, and apoptosis . RELA typically forms homo- or heterodimeric complexes with other NF-κB family proteins, with the p65-p50 heterodimer being the most abundant form in cells .

The biological significance of RELA stems from its role as an endpoint of multiple signal transduction pathways initiated by diverse stimuli. In its inactive state, RELA is sequestered in the cytoplasm by inhibitory proteins of the IκB family. Upon activation, IκB is phosphorylated by IκB kinases (IKKs), which leads to IκB degradation and liberation of active RELA complexes that translocate to the nucleus to regulate gene expression . This mechanism makes RELA a central player in numerous physiological and pathological processes, including inflammatory responses, cancer progression, and immune regulation.

How are RELA recombinant monoclonal antibodies generated?

RELA recombinant monoclonal antibodies are synthesized in vitro through a systematic process that offers advantages over traditional antibody production methods. The production typically follows these steps:

• Antibody gene isolation from B cells derived from immunoreactive rabbits or other sources

• Amplification of these genes through PCR techniques

• Cloning of amplified genes into phage vectors

• Introduction of vectors into mammalian cell lines (commonly Expi293F cells)

• Expression of antibodies by transfected cells

• Purification of secreted antibodies from culture supernatant through affinity chromatography

A more advanced approach involves epitope-directed antibody production, where in silico-predicted epitopes (13-24 residues long) are presented as three-copy inserts on surface-exposed loops of carrier proteins like thioredoxin. This method produces high-affinity antibodies reactive to both native and denatured RELA . The resulting recombinant antibodies offer improved standardization, reproducibility, and flexibility for customization compared to traditional hybridoma-derived antibodies.

What applications are RELA recombinant monoclonal antibodies suitable for?

RELA recombinant monoclonal antibodies are versatile tools suitable for multiple research applications. The table below outlines common applications and their associated parameters:

For optimal results in these applications, researchers should validate the antibody in their specific experimental system, optimize concentrations through titration experiments, and include appropriate positive and negative controls . The superior consistency of recombinant antibodies makes them particularly valuable for longitudinal studies where antibody performance must remain consistent across experiments.

How do I validate the specificity of RELA recombinant monoclonal antibodies?

Rigorous validation of RELA recombinant monoclonal antibodies is essential to ensure experimental reproducibility and data reliability. A comprehensive validation strategy should include:

• Genetic knockdown/knockout validation: Treat cells with RELA-specific siRNA or generate RELA knockout cell lines using CRISPR/Cas9. Compare antibody reactivity between control and RELA-depleted samples using immunofluorescence and immunoblotting. Specific antibodies will show significantly reduced signal in knockdown/knockout samples .

• Peptide competition assays: Pre-incubate the antibody with excess antigenic peptide before application to samples. Specific binding should be blocked in peptide-treated conditions while remaining intact in untreated controls.

• Multi-epitope validation: Use antibodies targeting different epitopes of RELA and confirm consistent results. This approach is particularly valuable for complex experiments where epitope accessibility might vary .

• Orthogonal method validation: Compare antibody-based detection with orthogonal techniques like mass spectrometry or RNA expression analysis to confirm protein identity and expression levels.

• Cross-reactivity assessment: Test the antibody against related family members (e.g., RELB, c-REL) to confirm specificity for RELA. This is particularly important for studying specific NF-κB pathway components.

Published studies demonstrate that cells depleted of target proteins show significantly reduced reactivity with properly validated recombinant antibodies in both cellular immunofluorescence and immunoblotting of cell lysates .

What advantages do RELA recombinant monoclonal antibodies offer over traditional antibodies?

RELA recombinant monoclonal antibodies provide several significant advantages over traditional hybridoma-derived antibodies:

• Enhanced reproducibility: The defined in vitro production process eliminates batch-to-batch variability inherent to traditional antibody production, addressing a major source of irreproducibility in research .

• Ethical considerations: Recombinant antibody production dramatically reduces animal use, addressing ethical concerns regarding the large number of animals traditionally used for antibody generation .

• Cost efficiency: While initial development costs may be higher, recombinant antibodies can be produced at lower costs for subsequent batches once the sequence is established .

• Customization potential: The recombinant platform allows for antibody engineering, including species specificity modification, isotype switching, and generation of various antibody fragments for specific applications .

• Epitope precision: The epitope-directed approach allows targeting of specific regions of RELA, enabling precise studies of different functional domains or post-translational modifications .

• Improved sensitivity: Comparative studies have demonstrated that recombinant antibodies can show modestly higher sensitivity than their traditional counterparts in applications like immunofluorescence .

• Perpetual availability: Once sequenced and produced recombinantly, antibodies can be manufactured indefinitely without reliance on hybridomas, which can be lost over time .

How do different epitope-targeting strategies affect RELA antibody performance?

The choice of target epitope on the RELA protein significantly impacts antibody performance across different applications. Researchers should consider these epitope-specific effects when selecting antibodies:

For optimal experimental design, researchers should implement a strategic approach to epitope selection:

• For detecting total RELA regardless of activation state, target conserved regions within the RHD that are accessible in both native and denatured states.

• For studying RELA activation dynamics, use antibodies targeting phosphorylation sites like Ser536, which serve as markers for active RELA.

• For distinguishing between RELA and related family members, target non-conserved regions that differ from RELB and c-REL.

• For co-immunoprecipitation studies, select antibodies targeting epitopes that do not interfere with protein-protein interactions of interest.

The epitope-directed approach using short antigenic peptides (13-24 residues) presented on carrier proteins has proven effective for generating antibodies that recognize both native and denatured forms of target proteins , making it valuable for comprehensive RELA studies.

How can I customize RELA recombinant monoclonal antibodies for specific experimental needs?

Recombinant monoclonal antibodies against RELA can be engineered and customized to meet specific experimental requirements through several approaches:

• Species specificity modification: The variable regions can be engineered to recognize RELA from different species by identifying conserved and divergent regions across species and modifying the complementarity-determining regions (CDRs) accordingly. This enables cross-species studies while maintaining epitope specificity .

• Antibody format conversion: Full-length IgG antibodies can be converted into smaller fragments (Fab, F(ab')2, scFv) to improve tissue penetration or reduce non-specific binding. Conversely, single-chain antibody fragments can be converted into full-length, bivalent antibodies to increase avidity, as demonstrated in studies where variable regions from scFv antibodies were successfully cloned onto IgG constant regions to create functional bivalent antibodies .

• Isotype switching: The constant regions can be exchanged to alter effector functions or compatibility with detection systems without affecting epitope recognition. For example, switching from mouse IgG to rabbit IgG enables use in multi-labeling experiments with mouse antibodies without cross-reactivity issues.

• Direct conjugation: Recombinant antibodies can be site-specifically conjugated with detection moieties (fluorophores, enzymes, affinity tags) to eliminate the need for secondary antibodies, reducing background and enabling multiplex studies.

• Affinity maturation: Directed evolution techniques can improve binding affinity through iterative rounds of mutation and selection in the CDR regions, enhancing sensitivity for detecting low-abundance RELA.

These customization approaches leverage the defined sequence and modular nature of recombinant antibodies to create tailored reagents for specific research applications .

How do post-translational modifications of RELA affect antibody recognition?

Post-translational modifications (PTMs) of RELA can significantly impact antibody recognition in ways that researchers must carefully consider when interpreting experimental results:

• Phosphorylation effects: RELA undergoes phosphorylation at multiple sites that regulate its activity and function. Key sites include:

  Phosphorylation Site	Functional Role	Impact on Antibody Recognition
  Ser536	Key activation marker	May create or mask epitopes
  Ser276	Regulates DNA binding	Can alter protein conformation
  Ser311	Affects transcriptional activity	May influence epitope accessibility

  Methodological approach: Use phospho-specific antibodies in parallel with total RELA antibodies to distinguish activation states. Treat samples with phosphatases to confirm phosphorylation-dependent recognition.

• Acetylation considerations: Acetylation of lysine residues in RELA affects DNA binding and transcriptional activity. Antibodies targeting these regions may show differential binding depending on acetylation status.

• Ubiquitination interference: Ubiquitination of RELA targets it for degradation and can physically block epitope accessibility, potentially leading to underestimation of RELA levels.

• Conformational changes: PTMs can induce significant conformational changes in RELA that expose or mask epitopes without directly modifying the antibody binding site itself.

For accurate experimental interpretation, researchers should:

• Use multiple antibodies targeting different RELA regions to create a comprehensive profile

• Include appropriate controls that account for different modification states

• Consider the biological context and likely modification status of RELA in their experimental system

• Validate findings with complementary approaches that are less sensitive to PTM status

The search results highlight that NF-κB (including RELA) is "controlled by various mechanisms of post-translational modification" , making this an important consideration for antibody selection and experimental design.

What strategies can resolve conflicting data obtained with different RELA antibodies?

When faced with conflicting results from different RELA antibodies, researchers should implement a systematic approach to resolve discrepancies:

• Epitope mapping analysis:

  • Determine the precise epitopes recognized by each antibody

  • Assess whether conflicts might stem from detection of different RELA isoforms or post-translationally modified variants

  • Consider epitope masking due to protein-protein interactions or conformational states

• Comprehensive antibody validation:

  • Perform side-by-side testing with RELA knockdown/knockout controls for each antibody

  • Include competing antigens to assess cross-reactivity profiles

  • Test antibodies against recombinant RELA alongside related family members (RELB, c-REL)

• Cross-validation with orthogonal methods:

  • Employ non-antibody-based detection methods (e.g., mass spectrometry)

  • Use genetic approaches (e.g., tagged RELA expression in knockout background)

  • Implement functional assays (e.g., reporter assays for RELA transcriptional activity)

• Multi-antibody consensus approach:

  • Test multiple antibodies targeting different RELA epitopes

  • Apply a majority rule or scoring system weighted by validation status

  • Document epitope-specific limitations for transparent reporting

• Standardized experimental conditions:

  • Use identical sample preparation protocols when comparing antibodies

  • Process samples in parallel rather than sequentially

  • Standardize image acquisition and analysis parameters

The literature highlights a case where inadequate antibody characterization led to significant controversies in growth differentiation factor 11 (GDF11) research, where an antibody was later found to cross-react with a closely-related family member, raising concerns about the validity of original findings . This underscores the importance of thorough validation and using multiple approaches to resolve conflicting data.

What are optimal conditions for using RELA recombinant monoclonal antibodies in different cell types?

Optimizing experimental conditions for RELA recombinant monoclonal antibodies across different cell types requires systematic adaptation of protocols. Based on research best practices, consider these methodological guidelines:

• Cell-type specific fixation protocols:

  Cell Type	Recommended Fixation	Incubation Time	Special Considerations
  Adherent epithelial cells	4% paraformaldehyde	10-15 minutes	Gentle permeabilization
  Suspension immune cells	2% paraformaldehyde	5-10 minutes	More stringent permeabilization
  Neural cells	2% paraformaldehyde + 0.1% glutaraldehyde	10 minutes	May require antigen retrieval
  Primary tissue	10% neutral buffered formalin	24-48 hours	Requires optimized antigen retrieval

• Antibody concentration optimization:

  • Perform titration experiments for each cell type (typically 0.1-10 μg/ml)

  • Compare signal-to-noise ratios across concentrations

  • Select optimal concentration that maximizes specific signal while minimizing background

  • Note that recombinant antibodies may demonstrate higher sensitivity than traditional antibodies in some applications

• Permeabilization method selection:

  • For cytoplasmic RELA detection: 0.1-0.5% Triton X-100 (5-15 minutes)

  • For nuclear RELA detection: 0.5% Triton X-100 or methanol permeabilization

  • For membrane-associated RELA: Consider saponin (0.1-0.5%) for reversible permeabilization

• Blocking strategy customization:

  • Cells with high Fc receptor expression: Include appropriate Fc blocking reagent

  • High autofluorescence samples: Consider Sudan Black B treatment

  • High background with traditional blocking: Test alternative blockers (fish gelatin, casein)

• Incubation time and temperature adjustment:

  • Primary antibody: 1-2 hours at room temperature or overnight at 4°C

  • Secondary antibody: 30-60 minutes at room temperature

  • Extend incubation times for thick tissue sections or poorly permeable samples

The literature indicates that antibody sensitivity can vary between recombinant and traditional versions, with some recombinant antibodies showing modestly higher sensitivity, which may influence optimal dilution determination .

How can RELA recombinant monoclonal antibodies be integrated into multiplexed detection systems?

Implementing RELA recombinant monoclonal antibodies in multiplexed detection systems requires careful consideration of several methodological factors:

• Species compatibility engineering:

  • Convert antibodies to different species formats (e.g., from mouse to rabbit) to enable co-staining with antibodies from other species

  • Validate absence of cross-reactivity with secondary antibodies used for other targets

  • Consider using directly conjugated primary antibodies to eliminate secondary antibody cross-reactivity entirely

• Epitope accessibility coordination:

  • Select antibodies targeting spatially distinct epitopes when co-staining for multiple proteins

  • Test sequential versus simultaneous application of antibodies to address potential steric hindrance

  • Validate signal intensity in single versus multiplex contexts to ensure detection sensitivity is maintained

• Signal separation optimization:

  • Choose fluorophores with minimal spectral overlap for immunofluorescence multiplexing

  • Implement computational unmixing for closely overlapping signals

  • Consider sequential chromogenic detection for brightfield applications

• Antibody format selection:

  • Use smaller antibody fragments (Fab, scFv) to reduce steric hindrance in densely labeled samples

  • Employ full-length IgG for targets requiring signal amplification

  • Generate species-switched versions of the same antibody for flexible experimental design

• Validation in multiplexed context:

  • Confirm antibody performance in the multiplex assay matches single-plex results

  • Include appropriate controls for each target in the multiplex panel

  • Validate specificity using genetic knockdown or knockout approaches for each target

The literature provides examples where researchers have successfully created species-specific antibodies by cloning variable regions from one species onto constant regions from another, specifically to enable multi-labeling experiments without cross-reactivity issues . This approach is particularly valuable for studying RELA in context with other NF-κB pathway components or downstream targets.

What are the latest methodological advances in generating high-specificity RELA recombinant monoclonal antibodies?

Recent technological advances have significantly improved the generation of high-specificity RELA recombinant monoclonal antibodies:

• Epitope-directed antibody production:
  A novel approach uses in silico-predicted epitopes presented as three-copy inserts on surface-exposed loops of thioredoxin carriers. This method produces high-affinity monoclonal antibodies reactive to both native and denatured forms of target proteins .

  Key advantages:

  • Enables targeting of multiple predicted epitopes in a single hybridoma production cycle

  • Facilitates direct epitope mapping crucial for antibody characterization

  • Allows generation of antibodies against spatially distant sites for validation purposes

• DEXT microplate technology:
  ELISA assay miniaturization using novel DEXT microplates enables rapid hybridoma screening with simultaneous epitope identification .

  Methodological impact:

  • Accelerates antibody screening process

  • Enables high-throughput epitope specificity determination

  • Reduces reagent consumption and increases efficiency

• Mixed antigen format approach:
  Using short antigenic peptides (13-24 residues long) as immunogens produces antibodies with high specificity and versatility across applications .

  Implementation strategy:

  • Select multiple epitopes from different domains of RELA

  • Present each as a defined peptide antigen

  • Generate a panel of complementary monoclonal antibodies

• Recombinant conversion of existing antibodies:
  For valuable existing hybridoma-derived antibodies, transcriptome sequencing can identify antibody sequences, enabling recombinant production with improved consistency .

  Technical approach:

  • Extract mRNA from hybridoma cells

  • Generate cDNA library and perform transcriptome sequencing

  • Identify antibody heavy and light chain sequences

  • Clone into expression vectors for recombinant production

• Format interconversion technologies:
  Methods to convert antibody fragments (scFv) into full-length antibodies and vice versa enable flexible experimental design .

  Practical application:

  • Extract variable region sequences from existing antibodies

  • Clone onto appropriate constant region frameworks

  • Express in mammalian cells for production of desired format

These advances address fundamental issues of antibody quality, validation, and utility that have contributed to irreproducibility in scientific research , providing researchers with more reliable tools for studying RELA biology.