p65 Antibody

What is p65 and what role does it play in cellular signaling?

P65 (also known as RelA) is a 65 kDa protein that functions as a subunit of the NF-κB transcription factor complex. It is encoded by the RELA gene in humans, with an accession number of Q04206 . NF-κB p65 plays a crucial role in regulating immune response, inflammation, cell proliferation, and apoptosis through its ability to control gene expression when activated.

The protein contains a Rel homology domain responsible for DNA binding, dimerization, and interaction with inhibitory proteins. When inactive, p65 is sequestered in the cytoplasm by inhibitory IκB proteins. Upon stimulation (such as by TNF-α), IκB becomes phosphorylated and degraded, allowing p65 to translocate to the nucleus where it regulates target gene expression . This dynamic translocation between cytoplasm and nucleus makes p65 an important subject in studies of cellular signaling mechanisms.

What are the key applications for p65 antibodies in research?

P65 antibodies are versatile tools employed across multiple research applications:

When selecting antibodies for specific applications, researchers should verify that the antibody has been validated for their particular experimental system and application .

How can I validate the specificity of p65 antibodies for my research?

Proper validation of p65 antibodies is critical for generating reliable data. Several approaches should be implemented:

• Knockout/knockdown controls: The most stringent validation method involves comparing antibody reactivity in wildtype versus p65 knockout or knockdown samples. A specific antibody will show absence of signal in knockout tissues, while nonspecific antibodies will continue to mark proteins in these negative control samples .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight (~65-70 kDa for p65) .

• Blocking peptide controls: While sometimes used, these can be insufficient for full validation. Some antibodies that pass blocking peptide tests may still fail specificity tests with knockout tissues .

• Multiple antibody comparison: Use several antibodies targeting different epitopes of p65 to verify consistent results.

• Positive controls: Include samples known to express p65 (e.g., K562, HeLa, or Daudi cell lines) .

• Treatment-induced changes: Verify expected changes in localization or phosphorylation following stimulation (e.g., TNF-α treatment should induce nuclear translocation) .

It's worth noting that some commercially available antibodies may exhibit nonspecific binding despite manufacturer claims, particularly those raised against the peptide sequence containing nuclear localization signals of p65 .

What factors should I consider when selecting between monoclonal and polyclonal p65 antibodies?

The choice between monoclonal and polyclonal p65 antibodies depends on your experimental goals:

For studies requiring precise detection of specific phosphorylated forms of p65, monoclonal antibodies targeting particular phosphorylation sites (e.g., serine 529) may be preferable . For general p65 detection across multiple species, affinity-purified polyclonal antibodies such as those recognizing human, mouse, and rat p65 might be more appropriate .

How can I simultaneously assess p65 nuclear translocation and phosphorylation status?

Simultaneous assessment of p65 nuclear translocation and phosphorylation provides comprehensive insights into NF-κB signaling dynamics. One effective approach employs imaging flow cytometry:

This approach allows researchers to correlate phosphorylation with translocation at the single-cell level, revealing potential subpopulations with distinct signaling characteristics. The technique has demonstrated reproducibility in detecting PMA/Ionomycin-induced changes in both p65 localization and phosphorylation .

What are the most reliable approaches for troubleshooting non-specific binding with p65 antibodies?

Non-specific binding is a common challenge with p65 antibodies, particularly in neuronal tissues where endogenous p65 levels may be low. Comprehensive troubleshooting includes:

• Antibody validation with knockout controls: This remains the gold standard for confirming antibody specificity. Even antibodies showing a single band at the correct molecular weight should be tested against knockout tissues .

• Optimization of blocking conditions:

  • Extend blocking time (up to 2 hours)

  • Test different blocking agents (BSA, normal serum, commercial blocking buffers)

  • Include 0.1-0.3% Triton X-100 in blocking solutions for better penetration

• Titration experiments: Perform detailed titration series to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Cross-adsorption: For polyclonal antibodies, cross-adsorption against tissues from knockout animals can reduce non-specific binding.

• Alternative detection methods: If one application (e.g., IHC) shows non-specificity, try alternative approaches like Western blot or flow cytometry.

• Secondary antibody controls: Always include controls omitting primary antibody to assess secondary antibody non-specific binding.

Importantly, some antibodies may perform well in Western blots but fail in immunohistochemistry. For example, antibodies raised against the peptide sequence containing the nuclear localization signals of p65 (such as MAB3026 from Chemicon and sc-114 from Santa Cruz) have shown particularly problematic non-specific binding .

How do phospho-specific p65 antibodies differ in their applications and limitations?

Phospho-specific p65 antibodies detect distinct phosphorylation events that regulate p65 function:

When working with phospho-specific antibodies:

• Phosphatase inhibitors are essential during sample preparation to preserve phosphorylation status.

• Fixation methods significantly impact epitope preservation; paraformaldehyde is generally preferred for phospho-epitopes.

• Signal amplification may be necessary for detecting low-abundance phosphorylated forms.

• Quantification challenges arise from the dynamic nature of phosphorylation; treatment durations and conditions must be carefully standardized.

• Validation approaches should include phosphatase treatment controls and stimulation with known activators like TNF-α or PMA/Ionomycin .

Researchers should be particularly cautious with antibodies against p65 phosphorylated at serine 276, as multiple commercial sources have shown problems with specificity despite passing blocking peptide tests .

What are the critical controls for p65 antibody applications in different experimental systems?

Rigorous controls are essential for meaningful p65 antibody experiments:

• For Western blotting:

  • Positive controls: Include lysates from cells known to express p65 (K562, Daudi, LNCaP, HeLa)

  • Negative controls: Include lysates from p65 knockout cells/tissues

  • Loading controls: Verify equal protein loading with housekeeping proteins

  • Molecular weight markers: Confirm correct size (approximately 65-70 kDa)

• For immunohistochemistry/immunofluorescence:

  • Tissue-specific controls: Include tissues known to express p65

  • Subcellular localization controls: Compare unstimulated (cytoplasmic) versus stimulated (nuclear) samples

  • Peptide competition: Pre-incubate antibody with immunizing peptide (with caution regarding interpretation)

  • Antibody omission: Control for non-specific binding of detection systems

• For monitoring activation:

  • Time course controls: Include multiple time points after stimulation

  • Positive activation control: TNF-α treatment (20 ng/mL) typically induces robust nuclear translocation within 10 minutes

  • Inhibitor controls: Include NF-κB pathway inhibitors to confirm specificity of activation response

• For cross-species applications:

  • Species-appropriate positive controls: Verify reactivity in relevant species (e.g., human, mouse, rat cell lines)

  • Epitope conservation analysis: Check sequence homology of the target epitope across species

Using appropriate combinations of these controls is essential for generating reliable and interpretable data across experimental systems.

What advanced quantification methods can be used to analyze p65 activation in single cells?

Single-cell analysis of p65 activation provides insights into heterogeneous responses within cell populations:

• Imaging flow cytometry: This technique combines the statistical power of flow cytometry with the spatial resolution of microscopy.

  • Allows quantification of nuclear translocation using similarity scores between nuclear dye and p65 staining

  • Enables simultaneous assessment of phosphorylation and translocation

  • Can process thousands of cells for robust statistical analysis

• High-content imaging:

  • Automated microscopy platforms quantify nuclear/cytoplasmic ratios of p65

  • Machine learning algorithms can classify cells based on activation status

  • Permits temporal analysis in fixed or live cells

• Live-cell imaging with fluorescent fusion proteins:

  • p65-GFP fusion constructs enable real-time visualization of translocation

  • Photoactivatable or photoconvertible tags allow pulse-chase experiments

  • FRET-based reporters can detect protein-protein interactions or conformational changes

• Single-cell sequencing integration:

  • Combine protein measurements (e.g., CyTOF or CITE-seq) with transcriptome analysis

  • Correlate p65 activation with downstream gene expression signatures

  • Identify novel regulatory relationships through computational analysis

• Quantitative image analysis parameters:

  • Nuclear/cytoplasmic ratio of p65 intensity

  • Mean fluorescence intensity of phospho-p65

  • Colocalization coefficients (Pearson's, Mander's) for multi-protein complexes

  • Tracking of oscillatory patterns in NF-κB signaling

These approaches should be validated using appropriate stimuli (like TNF-α) that induce well-characterized changes in p65 localization from cytoplasmic to nuclear compartments .

What are the optimal fixation and permeabilization methods for p65 antibody staining?

The choice of fixation and permeabilization methods significantly impacts p65 antibody staining quality:

For optimal permeabilization:

• For immunocytochemistry: 0.1% Triton X-100 in PBS provides adequate permeabilization for accessing nuclear p65

• For tissue sections: Higher detergent concentrations (0.2-0.3% Triton X-100) may be necessary

• For flow cytometry: Saponin-based buffers (0.1%) offer gentler permeabilization with good epitope preservation

When detecting phosphorylated p65, always include phosphatase inhibitors in all buffers and minimize processing time to preserve phosphorylation status . For dual staining applications, a sequential approach may be necessary, with phospho-specific antibodies applied first.

How should I optimize Western blot protocols for detecting p65 and its modified forms?

Western blot optimization for p65 detection requires attention to several critical factors:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • For phosphorylated p65, add phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Fractionate samples (cytoplasmic vs. nuclear) when studying translocation

• Gel percentage and running conditions:

  • 8-10% SDS-PAGE gels are optimal for resolving p65 (~65-70 kDa)

  • Ensure adequate separation from potential cross-reactive proteins

• Transfer conditions:

  • Semi-dry or wet transfer systems both work well

  • Transfer at lower voltage for longer times to ensure complete transfer of larger proteins

• Blocking:

  • 5% non-fat dry milk in TBST is standard, but may not be optimal for phospho-specific antibodies

  • For phospho-p65 detection, use 5% BSA in TBST instead of milk (phospho-epitopes can bind to casein)

• Antibody dilution and incubation:

  • Typical dilutions range from 1:200 to 1:1000 depending on the antibody

  • Affinity-purified antibodies may require lower dilutions (1 μg/mL)

  • Overnight incubation at 4°C often yields better results than short incubations

• Detection system:

  • HRP-conjugated secondary antibodies work well for most applications

  • For low abundance targets, consider enhanced chemiluminescence or fluorescent secondaries

• Controls and troubleshooting:

  • Include positive control lysates (K562, HeLa, or Daudi cell lines)

  • Run parallel blots with total and phospho-specific antibodies

  • Use anti-histone and anti-tubulin antibodies to verify fractionation quality

These optimizations help ensure specific detection of p65 and its modified forms while minimizing background and non-specific signals.

What are the best approaches for quantifying p65 nuclear translocation?

Accurate quantification of p65 nuclear translocation provides crucial information about NF-κB pathway activation:

• Subcellular fractionation and Western blotting:

  • Separate nuclear and cytoplasmic fractions using commercial kits or differential centrifugation

  • Perform Western blots on both fractions

  • Calculate the nuclear/cytoplasmic ratio after normalization to loading controls (lamin for nuclear, tubulin for cytoplasmic)

  • Advantage: Biochemical quantification of large cell populations

  • Limitation: Loses single-cell information and may introduce fractionation artifacts

• Immunofluorescence microscopy with image analysis:

  • Perform dual staining for p65 and nuclear marker (DAPI)

  • Capture images at consistent exposure settings

  • Use image analysis software to:

    • Define nuclear and cytoplasmic regions

    • Measure mean fluorescence intensity in each compartment

    • Calculate nuclear/cytoplasmic ratio for multiple cells

  • Advantage: Preserves single-cell data and morphological context

  • Limitation: Lower throughput than flow-based methods

• Imaging flow cytometry with similarity scoring:

  • Label cells with nuclear dye and p65 antibodies

  • Calculate similarity scores between nuclear and p65 images

  • Higher similarity scores indicate greater nuclear localization

  • Plot distributions of similarity scores across cell populations

  • Advantage: High-throughput single-cell analysis with statistical power

  • Example: PMA/Ionomycin stimulation shifts median similarity scores to higher values, indicating nuclear translocation

• Automated high-content screening:

  • Analyze thousands of cells in multi-well format

  • Apply machine learning algorithms for complex phenotype recognition

  • Track translocation kinetics with time-lapse imaging

  • Advantage: Compatible with drug screening applications

  • Limitation: Requires specialized equipment and expertise

The choice of method depends on experimental goals, available equipment, and whether single-cell resolution is required.

Why might my p65 antibody show different results across applications?

Inconsistent performance across applications is a common challenge with p65 antibodies:

• Epitope accessibility differences:

  • Some epitopes may be masked in certain applications but exposed in others

  • Fixed tissues may exhibit different epitope presentation than denatured proteins in Western blots

  • Studies show that antibodies can perform well in Western blots but fail in immunohistochemistry

• Application-specific technical factors:

  • Western blotting uses denatured proteins, while IHC/IF typically preserve native conformation

  • Fixation methods alter protein structure differently (cross-linking vs. precipitating fixatives)

  • Buffer compositions affect antibody-antigen interactions

• Context-dependent expression levels:

  • Low endogenous p65 levels in some tissues may be below detection threshold for certain applications

  • Background signal can overwhelm specific signal in tissues with low expression

• Antibody characteristics affecting performance:

  • Antibody affinity and avidity differences impact performance across applications

  • Polyclonal antibodies may recognize different epitopes with varying accessibility

  • Clone-specific characteristics of monoclonal antibodies can limit cross-application utility

To address these challenges:

• Validate each antibody specifically for your application of interest

• Use antibodies that have been validated in knockout tissues for your specific application

• Consider using multiple antibodies targeting different epitopes

• Optimize protocols specifically for each application rather than using standardized conditions

How can I minimize batch-to-batch variability when working with p65 antibodies?

Batch-to-batch variability can significantly impact experimental reproducibility:

• Antibody selection strategies:

  • Monoclonal antibodies generally show less batch variability than polyclonals

  • Recombinant antibodies offer greater consistency than hybridoma or serum-derived antibodies

  • Affinity-purified antibodies typically show better consistency than crude antisera

• Validation for each new batch:

  • Always perform side-by-side comparison with previous batches

  • Test new batches on consistent positive control samples

  • Verify expected molecular weight and staining pattern

  • Document lot numbers and include in publications

• Standardization practices:

  • Create standard operating procedures (SOPs) for each application

  • Maintain consistent sample preparation methods

  • Use automated systems where possible to reduce technical variability

  • Implement quality control checkpoints throughout protocols

• Long-term planning:

  • Purchase larger amounts of validated antibodies when possible

  • Aliquot and store according to manufacturer recommendations to avoid freeze-thaw cycles

  • Consider establishing collaborations with colleagues to share validated antibody batches

  • Document performance characteristics of different batches for long-term studies

• Quantitative approaches:

  • Include standard curves or reference samples in each experiment

  • Normalize results to consistent internal controls

  • Use ratiometric measurements rather than absolute values when possible

These strategies help minimize the impact of batch variability on experimental outcomes and improve reproducibility across studies.

What are the common pitfalls when detecting phosphorylated forms of p65?

Detection of phosphorylated p65 presents unique challenges:

• Rapid dephosphorylation:

  • Phosphorylated epitopes are highly labile

  • Always use fresh phosphatase inhibitors in all buffers

  • Minimize processing time between sample collection and fixation/lysis

  • Keep samples cold during processing to slow enzymatic activity

• Epitope masking:

  • Phosphorylation-specific conformational changes can affect epitope accessibility

  • Some fixation methods may preserve phosphorylation but mask epitopes

  • Consider multiple fixation approaches if initial attempts fail

• Antibody specificity issues:

  • Many phospho-specific antibodies show cross-reactivity with non-phosphorylated epitopes

  • Antibodies against p65 phosphorylated at serine 276 have shown particular specificity problems

  • Blocking peptide validation alone is insufficient; knockout/knockdown controls are essential

• Signal-to-noise challenges:

  • Phosphorylated forms often represent a small fraction of total protein

  • Background signal can easily overwhelm specific signal

  • Signal amplification techniques may be necessary but can introduce artifacts

• Treatment-dependent effects:

  • Phosphorylation is dynamic and stimulation-dependent

  • Inconsistent stimulation protocols lead to variable results

  • Different cell types show different phosphorylation kinetics

  • Always include positive controls (TNF-α treated samples)

To address these challenges:

• Use lambda phosphatase-treated control samples to verify phospho-specificity

• Apply directly labeled phospho-specific antibodies to reduce processing steps

• Consider alternative approaches like Phos-tag gels for biochemical validation

• Implement careful time-course experiments to capture transient phosphorylation events

How should I approach species cross-reactivity when using p65 antibodies?

Cross-species applications require careful consideration:

• Epitope conservation analysis:

  • Compare sequence homology of target epitopes across species

  • Higher conservation in the epitope region suggests better cross-reactivity

  • The C-terminal region of p65 (amino acids 456-551) shows good conservation across human, mouse, and rat

• Validation strategies for cross-species use:

  • Test antibodies on known positive samples from each species

  • Include appropriate negative controls (knockout/knockdown samples when possible)

  • Verify consistent band size across species (accounting for species-specific variations)

• Application-specific considerations:

  • Western blotting often shows better cross-reactivity than IHC/IF

  • Species-specific secondary antibodies are essential for avoiding background

  • Tissue-specific factors may affect accessibility regardless of sequence homology

• Documented cross-reactivity:

  • Some antibodies are specifically validated for multiple species:

    • Anti-Human/Mouse/Rat RelA/NF kappa B p65 antibody (AF5078) shows reactivity in human (K562, HeLa), mouse (Neuro-2A), and rat (C6) cell lines

    • NFKB p65 Polyclonal Antibody from Bioss shows reactivity across human, mouse, rabbit, rat, bovine, chicken, and zebrafish

• Optimization for new species:

  • Titrate antibody concentrations when testing in new species

  • Modify blocking conditions to account for species-specific background

  • Consider species-specific sample preparation modifications

When selecting antibodies for cross-species applications, prioritize those with documented validation in your target species rather than relying on predicted cross-reactivity based on sequence homology alone.

How can multiplexed approaches be used to study p65 in complex signaling networks?

Modern multiplexed technologies enable simultaneous analysis of p65 alongside other signaling components:

• Multiplex immunofluorescence/immunohistochemistry:

  • Sequential staining protocols allow detection of 5-10 targets on the same sample

  • Tyramide signal amplification enables using multiple primary antibodies from the same species

  • Spectral unmixing algorithms separate overlapping fluorophore signals

  • Applications: Simultaneously detect p65, other NF-κB subunits, upstream kinases, and downstream targets

• Mass cytometry (CyTOF):

  • Metal-tagged antibodies enable detection of 40+ parameters simultaneously

  • No spectral overlap concerns, unlike fluorescence-based approaches

  • Lower sensitivity than fluorescence but excellent for abundant proteins like p65

  • Applications: Map p65 activation across cell subsets in heterogeneous samples

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions or modified proteins with high specificity

  • Signal amplification allows detection of low-abundance complexes

  • Applications: Study interactions between p65 and binding partners or detect specific phosphorylated forms

• Single-cell proteogenomics:

  • CITE-seq or REAP-seq combine protein measurement with transcriptomics

  • Correlate p65 protein levels/modifications with gene expression signatures

  • Applications: Link signaling states to transcriptional outcomes in individual cells

• Spatial proteomics:

  • Imaging mass cytometry or Codex enable spatial mapping of dozens of proteins

  • Maintain tissue architecture context while assessing multiple markers

  • Applications: Study p65 activation patterns in complex tissues like tumors or brain

These multiplexed approaches provide richer context for understanding p65 function within broader signaling networks, enabling systems-level analysis of NF-κB pathway regulation.

What considerations are important when using p65 antibodies in challenging sample types?

Certain sample types present unique challenges for p65 antibody applications:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Epitope retrieval is critical: Test multiple methods (heat-induced vs. enzymatic)

  • Extended retrieval times may be necessary for heavily fixed samples

  • Polyclonal antibodies often perform better than monoclonals in FFPE material

  • Example: Anti-Human/Mouse/Rat RelA/NF kappa B p65 antibody has been validated for FFPE human squamous cell carcinoma

• Brain and neuronal samples:

  • Higher background and non-specific binding in neuronal tissues

  • Low endogenous p65 levels can be difficult to detect above background

  • Multiple p65 antibodies show non-specific binding in neurons despite manufacturer claims

  • Recommendations: Use knockout controls, optimize blocking, consider alternative detection methods

• Primary cell isolates:

  • Limited material requires sensitive detection methods

  • Higher variability compared to cell lines necessitates more replicates

  • Donor-specific differences can impact baseline and stimulated p65 levels

  • Recommendations: Pool samples when possible, implement robust normalization

• Clinical specimens:

  • Pre-analytical variables (collection, fixation, storage) significantly impact results

  • Standardized protocols are essential for reproducible results

  • Batch effects require careful experimental design

  • Recommendations: Include technical controls, process all comparative samples simultaneously

• Archival samples:

  • Antigen degradation over time affects detection sensitivity

  • Fixation artifacts become more pronounced in older samples

  • Recommendations: Include freshly processed controls, adjust exposure/development times