RELA (Ab-311) Antibody

What is RELA (Ab-311) antibody and what epitope does it target?

RELA (Ab-311) antibody is a polyclonal antibody produced in rabbits that specifically recognizes the human RELA protein (also known as p65, NFKB3, or NF-κB p65 subunit). This antibody targets a synthetic non-phosphopeptide derived from NF-κB p65 around the phosphorylation site of serine 311, specifically recognizing the amino acid sequence F-K-S-I-M . It functions as an affinity isolated antibody with an approximate molecular weight of 65 kDa and belongs to the IgG isotype class . RELA is a critical component of the NF-κB transcription factor complex involved in numerous cellular processes including inflammation, immunity, cell differentiation, growth, and apoptosis .

What species reactivity does RELA (Ab-311) antibody demonstrate?

RELA (Ab-311) antibody has been validated for recognizing RELA proteins from multiple species including human, mouse, and rat samples . This cross-species reactivity makes it a versatile tool for comparative studies examining NF-κB signaling across different model organisms. Researchers should note that while the antibody demonstrates robust cross-reactivity, validation experiments should still be performed when using it in less common species or specialized tissue types to confirm specificity.

What are the recommended dilutions for RELA (Ab-311) antibody in common applications?

Based on validation studies, the following application-specific dilutions are recommended:

Each new lot should be titrated to determine optimal concentration for your specific experimental conditions. Starting with the middle of the recommended range is advisable for initial optimization.

What are the proper storage conditions for maintaining RELA (Ab-311) antibody activity?

For optimal antibody performance and longevity, RELA (Ab-311) antibody should be stored at -20°C . The product is typically shipped on wet ice and should be immediately transferred to -20°C upon receipt. For working solutions, aliquoting is strongly recommended to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly reduce antibody activity and specificity. Each aliquot should be sufficient for a single experiment to avoid the need for refreezing. Avoid prolonged exposure to light and heat during experimental procedures.

How should I design controls for experiments using RELA (Ab-311) antibody?

A robust experimental design when using RELA (Ab-311) antibody should include the following controls:

• Positive Control: Include a cell line or tissue sample known to express RELA/p65 (most mammalian cell lines express detectable levels)

• Negative Control: Consider using:

  • Primary antibody omission control

  • Non-immune rabbit IgG at the same concentration

  • RELA/p65 knockout or knockdown samples (if available)

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (F-K-S-I-M) to confirm specificity

• Phosphorylation State Controls: If studying S311 phosphorylation effects, include samples treated with phosphatase inhibitors and phosphatase enzymes

These controls help validate antibody specificity and distinguish true signal from background, especially important when studying modifications around the S311 site.

What cell stimulation protocols are recommended for studying NF-κB activation with RELA (Ab-311) antibody?

When designing experiments to study NF-κB activation using RELA (Ab-311) antibody, consider the following established stimulation protocols:

For studying the specific epitope recognized by Ab-311 (around S311), treatments that induce phosphorylation at this site are particularly relevant. The S311 site is predominantly phosphorylated by PKCζ, so PKC activators may provide useful experimental conditions. Time-course experiments are recommended as NF-κB activation is typically transient and may vary between cell types.

How can I optimize Western blot protocols for RELA (Ab-311) antibody?

For optimal Western blot results with RELA (Ab-311) antibody, consider these technical recommendations:

• Sample Preparation:

  • Include phosphatase inhibitors in lysis buffer if studying phosphorylation states

  • For nuclear translocation studies, perform nuclear/cytoplasmic fractionation

  • Use 20-40 μg of total protein per lane

• Gel/Transfer Conditions:

  • 10% SDS-PAGE gels work well for resolving the ~65 kDa RELA protein

  • PVDF membranes are preferred over nitrocellulose for phosphoprotein detection

• Blocking/Antibody Incubation:

  • 5% BSA in TBST is recommended (preferred over milk for phosphoprotein studies)

  • Primary antibody incubation: 1:500-1:1000 dilution overnight at 4°C

  • Secondary antibody: Anti-rabbit HRP at 1:5000-1:10000 for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence with short (1-5 min) exposure times typically works well

  • Expected band size is approximately 65 kDa

If multiple bands appear, further optimization of antibody concentration or blocking conditions may be necessary.

Why might I observe multiple bands when using RELA (Ab-311) antibody in Western blot?

Multiple bands when using RELA (Ab-311) antibody could result from several factors:

• Post-translational Modifications: RELA/p65 undergoes numerous modifications including phosphorylation, acetylation, methylation, and ubiquitination, which can alter migration patterns. The Ab-311 antibody recognizes a region around S311, which is a key phosphorylation site.

• Proteolytic Degradation: NF-κB proteins can undergo partial degradation during sample preparation. Ensure protease inhibitors are fresh and used at appropriate concentrations.

• Splice Variants: Several isoforms of RELA have been documented, including p65Δ3, which could appear as distinct bands.

• Cross-Reactivity: Although the antibody is specific for RELA, high concentrations may detect related proteins with similar epitopes in the Rel family.

To address these issues:

• Use freshly prepared samples with complete protease inhibitor cocktails

• Include phosphatase inhibitors if studying phosphorylation states

• Optimize antibody concentration (try more diluted solutions)

• Consider peptide competition assays to confirm specificity

How can I address high background issues in immunohistochemistry with RELA (Ab-311) antibody?

High background in IHC when using RELA (Ab-311) antibody can be addressed through these optimization steps:

• Antibody Dilution: Start with more dilute antibody preparations (1:100-1:200) than recommended

• Blocking Optimization:

  • Extend blocking time to 1-2 hours at room temperature

  • Try different blocking agents: 5-10% normal goat serum, 1-3% BSA, or commercial blocking reagents

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

• Antigen Retrieval Adjustment:

  • Compare heat-induced epitope retrieval methods (citrate pH 6.0 vs. EDTA pH 9.0)

  • Optimize retrieval duration (10-30 minutes)

• Washing Steps:

  • Increase washing time and buffer volume

  • Add 0.05-0.1% Tween-20 to wash buffers

• Endogenous Peroxidase/Biotin Blocking:

  • For peroxidase-based detection, ensure complete quenching of endogenous peroxidase

  • For biotin-based systems, include avidin/biotin blocking steps

Compare results with no-primary antibody controls to distinguish between primary antibody-specific and secondary antibody-related background.

What approaches can address inconsistent RELA (Ab-311) antibody performance between experiments?

Inconsistent performance between experiments can significantly impact research reliability. Consider these approaches:

• Antibody Storage and Handling:

  • Aliquot antibody upon receipt to minimize freeze-thaw cycles

  • Maintain consistent storage at -20°C

  • Check for signs of antibody deterioration (precipitates, cloudiness)

• Protocol Standardization:

  • Document exact conditions for successful experiments

  • Create detailed step-by-step protocols with timing, temperature, and buffer compositions

  • Use the same lot of secondary antibodies and detection reagents

• Sample Preparation Consistency:

  • Standardize cell culture conditions (passage number, confluence)

  • Use consistent lysis procedures and buffer compositions

  • Process all experimental samples simultaneously when possible

• Quantitative Controls:

  • Include internal loading controls for Western blots

  • Run standard curves with known positive samples

  • Consider using recombinant RELA protein as a reference standard

• Antibody Validation:

  • Test each new lot against previous lots that performed well

  • Consider using alternative antibodies targeting different RELA epitopes for confirmation

Implementing these practices can significantly improve reproducibility across experiments.

How can RELA (Ab-311) antibody be utilized in studying NF-κB signaling dynamics?

RELA (Ab-311) antibody can be employed in several advanced applications to study NF-κB signaling dynamics:

• Chromatin Immunoprecipitation (ChIP):

  • Use at 4-5 μg per immunoprecipitation to analyze RELA binding to specific gene promoters

  • Particularly useful for studying how S311 phosphorylation affects DNA binding capabilities

• Proximity Ligation Assay (PLA):

  • Combine with antibodies against other NF-κB components or transcriptional cofactors

  • Allows visualization of protein-protein interactions in situ

  • Can detect transient interactions during signaling events

• Live Cell Imaging:

  • When conjugated to cell-permeable carriers or used with transiently permeabilized cells

  • Enables tracking of RELA nuclear translocation kinetics in real-time

• Phospho-specific Signaling Analysis:

  • The epitope near S311 makes this antibody valuable for examining PKC-mediated NF-κB regulation

  • Can be used in parallel with phospho-S311-specific antibodies to determine modification status

• Single-cell Analysis:

  • Compatible with flow cytometry (intracellular staining) for quantifying RELA expression/localization at single-cell resolution

  • Useful for identifying cellular heterogeneity in responses to NF-κB-activating stimuli

These applications enable researchers to examine both spatial and temporal aspects of NF-κB signaling with high precision.

What are the considerations for using RELA (Ab-311) antibody in multiplexed assays?

When incorporating RELA (Ab-311) antibody into multiplexed detection systems, consider these important factors:

• Antibody Species and Isotype Compatibility:

  • Being a rabbit polyclonal IgG, it pairs well with mouse monoclonals in dual staining

  • Use secondary antibodies with minimal cross-reactivity

  • Consider species-specific secondary antibodies (anti-rabbit that doesn't recognize mouse IgG)

• Spectral Overlap Management:

  • Choose fluorophores with minimal spectral overlap when designing immunofluorescence panels

  • Include single-stained controls for compensation in flow cytometry or spectral imaging

• Sequential Staining Protocols:

  • For co-localization with other rabbit antibodies, consider sequential immunostaining with direct labeling

  • Try tyramide signal amplification (TSA) to allow antibody stripping and re-probing

• Epitope Accessibility:

  • The Ab-311 epitope near S311 may be masked in certain protein complexes

  • Consider mild fixation conditions or native protein analysis when possible

• Antibody Concentration Balancing:

  • Titrate RELA (Ab-311) antibody in the context of other antibodies in the panel

  • Dominant signals may require reducing antibody concentration to achieve balanced detection

Proper controls, including FMO (fluorescence minus one) for flow cytometry applications, are essential for accurate interpretation of multiplexed data.

How can RELA (Ab-311) antibody be used to investigate cross-talk between NF-κB and other signaling pathways?

RELA (Ab-311) antibody can be strategically employed to investigate signaling cross-talk through these approaches:

• Co-immunoprecipitation Studies:

  • Use RELA (Ab-311) as the capture antibody to pull down associated proteins

  • Identify novel interaction partners through mass spectrometry

  • Verify specific interactions with candidate pathway components

• Pathway Inhibitor Experiments:

  • Combine with inhibitors of intersecting pathways (MAPK, JAK/STAT, PI3K/AKT)

  • Monitor changes in RELA phosphorylation, localization, or DNA binding

  • Create pathway inhibition matrices to map signal integration

• Post-translational Modification Analysis:

  • Use in tandem with antibodies against specific modifications (phosphorylation, acetylation)

  • The S311 region is particularly relevant for PKC-mediated regulation

  • Compare modification patterns across different stimulation conditions

• Transcriptional Reporter Assays:

  • Combine with luciferase or fluorescent reporters to measure functional outcomes

  • Correlate RELA binding/modification with transcriptional activity

  • Use siRNA/shRNA approaches to validate pathway components

• Spatial Colocalization Analysis:

  • Dual immunofluorescence with markers of other pathways

  • Quantify nuclear co-localization with transcription factors from other pathways

  • Track co-localization dynamics following various stimuli

These approaches can reveal mechanisms of pathway integration and signal prioritization in complex cellular responses.

What are the considerations for using RELA (Ab-311) antibody in cancer research models?

When employing RELA (Ab-311) antibody in cancer research contexts, researchers should consider:

• Constitutive NF-κB Activation:

  • Many cancers show aberrant, constitutive NF-κB activation

  • Titrate antibody concentrations for each cancer model, as RELA expression can vary dramatically

  • Compare nuclear/cytoplasmic ratios between normal and malignant cells

• Mutation Analysis:

  • RELA mutations occur in certain cancers (particularly lymphomas)

  • Verify epitope integrity if studying cancers with potential RELA mutations near S311

  • Consider sequencing RELA in your model system if antibody shows unexpected patterns

• Pharmacological Interventions:

  • Use alongside NF-κB inhibitors to correlate biochemical effects with phenotypic outcomes

  • Particularly useful for examining mechanisms of drug resistance

  • Time-course studies can reveal dynamic adaptations to treatment

• Tumor Microenvironment:

  • Evaluate RELA expression/activation in both tumor and stromal compartments

  • Multiplex with cell-type specific markers to differentiate RELA activity in tumor vs. immune cells

  • Consider tissue clearing techniques for 3D tumor analysis

• Patient-derived Models:

  • Validate antibody performance in patient-derived xenografts and organoids

  • Correlate RELA activation patterns with clinical outcomes and treatment responses

The antibody's ability to recognize human, mouse, and rat RELA makes it particularly valuable for translational studies across model systems.

What methodological adaptations are needed when using RELA (Ab-311) antibody in difficult tissue types?

Certain tissues present challenges for antibody-based detection of RELA. Consider these methodological adaptations:

• Adipose Tissue:

  • Extended fixation times may be required

  • Modify dehydration protocols to ensure complete paraffin infiltration

  • Consider cryosectioning as an alternative to paraffin embedding

  • Extended permeabilization steps improve antibody penetration

• Brain Tissue:

  • Optimize antigen retrieval (extended EDTA pH 9.0 often works well)

  • Reduce autofluorescence with Sudan Black or TrueBlack treatments

  • Consider vibratome sectioning for improved epitope preservation

  • Increase antibody incubation times (up to 48-72 hours at 4°C for thick sections)

• Bone/Calcified Tissues:

  • EDTA decalcification preserves epitopes better than acid-based methods

  • Extend antigen retrieval times

  • Use tyramide signal amplification for enhanced sensitivity

  • Optimize protease-based antigen retrieval if heat-based methods fail

• Highly Fibrotic Tissues:

  • Increase detergent concentration in wash buffers

  • Consider adding carrier proteins to antibody dilution buffers

  • Try enzymatic pre-treatment (proteinase K or trypsin)

  • Extend antibody incubation times with gentle agitation

• Tissue Microarrays (TMAs):

  • Reduce antibody concentration compared to whole sections

  • Extend washing steps to reduce edge artifacts

  • Optimize blocking to prevent background in diverse tissue types

These adaptations significantly improve detection in challenging tissue contexts while maintaining specificity.

How can RELA (Ab-311) antibody contribute to understanding the role of NF-κB in inflammatory diseases?

RELA (Ab-311) antibody offers several valuable approaches for investigating NF-κB's role in inflammatory diseases:

• Biomarker Development:

  • Quantify nuclear RELA as a measure of pathway activation in patient biopsies

  • Correlate with disease severity scores and treatment responses

  • Develop IHC scoring systems for clinical application

• Therapeutic Target Validation:

  • Monitor RELA activation following treatment with anti-inflammatory agents

  • Identify cell populations resistant to NF-κB inhibition

  • Evaluate the RELA activation status in treatment non-responders

• Inflammation Models:

  • Track RELA nuclear translocation kinetics during disease progression

  • Combine with immune cell markers to identify key cellular drivers

  • Compare activation patterns across different inflammatory disease models

• Genetic Association Studies:

  • Examine how disease-associated polymorphisms affect RELA expression/activation

  • Correlate genotypes with RELA activation patterns

  • Useful for stratifying patient populations based on molecular phenotypes

• Resolution Phase Analysis:

  • Investigate RELA activation during inflammation resolution

  • Correlate with expression of resolution mediators

  • Time-course studies can reveal delayed resolution mechanisms

The antibody's ability to recognize the region around S311, a key regulatory phosphorylation site, makes it particularly useful for understanding how NF-κB activation is modulated in disease contexts.

What are the limitations of RELA (Ab-311) antibody that researchers should be aware of?

While RELA (Ab-311) antibody is a valuable research tool, researchers should be cognizant of these limitations:

• Epitope Specificity Considerations:

  • The antibody targets a region around S311, which may be affected by post-translational modifications

  • Other modifications near this epitope could potentially affect antibody binding

  • The polyclonal nature means lot-to-lot variability may occur

• Application Restrictions:

  • Limited validation for certain applications (e.g., ChIP-seq, super-resolution microscopy)

  • May not be optimal for detecting all RELA isoforms or splice variants

  • Performance in certain fixation conditions may vary

• Technical Challenges:

  • As with many transcription factor antibodies, sensitivity may be insufficient for detecting low expression levels

  • Nuclear localization can create challenges for adequate permeabilization

  • May require optimization beyond standard protocols in specialized applications

• Interpretation Complexities:

  • Cannot distinguish between active and inactive RELA based solely on detection

  • Nuclear localization is often used as a proxy for activation, but additional confirmatory assays are advised

  • Cross-reactivity with other Rel family members possible at high concentrations

What emerging technologies might enhance the utility of RELA (Ab-311) antibody in future research?

Several emerging technologies offer promising enhancements for RELA (Ab-311) antibody applications:

• Spatial Transcriptomics Integration:

  • Combining immunofluorescence using RELA (Ab-311) with spatial transcriptomics

  • Correlating RELA localization with downstream gene expression in tissue context

  • Enables linking protein-level observations with transcriptional outcomes

• Mass Cytometry (CyTOF) Applications:

  • Metal-conjugated RELA (Ab-311) for high-dimensional single-cell analysis

  • Simultaneous detection of dozens of other signaling proteins

  • Particularly valuable for complex immune cell phenotyping

• Microfluidic Live-Cell Analysis:

  • Antibody conjugation for live-cell immunofluorescence

  • Real-time monitoring of RELA dynamics in response to stimuli

  • Combined with microfluidic delivery of stimuli/inhibitors

• CRISPR-Based Tagging:

  • CRISPR knock-in of epitope tags near the Ab-311 target region

  • Enables live-cell studies with complementary detection methods

  • Can resolve potential specificity issues by providing orthogonal validation

• Machine Learning Image Analysis:

  • Automated quantification of RELA nuclear/cytoplasmic ratios

  • Pattern recognition across large tissue datasets

  • Correlation of subtle RELA distribution patterns with disease states

These technologies will expand the scope and resolution of NF-κB pathway analysis using the RELA (Ab-311) antibody in the coming years.

How might researchers combine RELA (Ab-311) antibody with systems biology approaches for more comprehensive pathway analysis?

Integrating RELA (Ab-311) antibody detection with systems biology approaches offers powerful new insights:

• Multi-omics Integration:

  • Combine RELA ChIP-seq/ChIP-qPCR with RNA-seq following stimulation/inhibition

  • Correlate RELA binding patterns with changes in phosphoproteome

  • Identify direct vs. indirect gene regulation through network analysis

• Pathway Modeling:

  • Use quantitative RELA data to constrain mathematical models of NF-κB signaling

  • Test predictions of pathway cross-talk through targeted perturbations

  • Refine models using time-resolved RELA activation data

• Single-cell Multi-parameter Analysis:

  • Combine RELA (Ab-311) with antibodies against other pathway components

  • Identify cell state-dependent variations in NF-κB signaling

  • Discover new cellular subtypes based on signaling patterns

• Genome-wide Screens:

  • Use RELA nuclear translocation as a readout for CRISPR or RNAi screens

  • Identify novel regulators of NF-κB signaling

  • Validate hits using RELA (Ab-311) antibody in secondary assays

• Longitudinal Patient Monitoring:

  • Standardized RELA activation assays for clinical samples

  • Track changes in pathway activation during disease progression

  • Correlate with treatment responses and clinical outcomes