Phospho-RELA (Ser468) Antibody

What is Phospho-RELA (Ser468) Antibody and what specifically does it detect?

Phospho-RELA (Ser468) Antibody is a rabbit-derived antibody that specifically recognizes the phosphorylated form of NF-κB p65 (RELA) at the Serine 468 residue. This antibody has been raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 468 of human NF-κB p65 and does not cross-react with other phosphorylation sites . The antibody detects endogenous levels of the phosphorylated protein, which has a molecular weight of approximately 65 kDa . Phosphorylation at Ser468 represents a specific regulatory event in NF-κB signaling that can influence transcriptional activity distinct from other phosphorylation sites.

Which species does Phospho-RELA (Ser468) Antibody react with?

The Phospho-RELA (Ser468) Antibody demonstrates reactivity across multiple species. According to validation studies, this antibody reliably detects phosphorylated RELA/NF-κB p65 at Ser468 in human, mouse, and rat samples . This cross-species reactivity makes it valuable for comparative studies and translational research where findings need to be verified across different model organisms.

What research applications is Phospho-RELA (Ser468) Antibody validated for?

This antibody has been validated for several key research applications:

• Western Blotting (WB): For detecting phosphorylated RELA in cell or tissue lysates

• Immunoprecipitation (IP): For enriching and isolating phosphorylated RELA from complex protein mixtures

• In-Cell ELISA: For quantifying phosphorylated RELA in fixed cells while preserving cellular architecture and context

These applications enable researchers to investigate phosphorylation events in various experimental contexts from protein-level detection to cellular localization.

How should cells be prepared for In-Cell ELISA using Phospho-RELA (Ser468) Antibody?

For optimal results with In-Cell ELISA using Phospho-RELA (Ser468) Antibody, follow these methodological steps:

For adherent cells:

• Seed cells in 96-well plates to achieve approximately 80% confluence at the time of fixing

• Treat cells according to your experimental design

• Fix cells by replacing growth medium with 100 μL of 4% formaldehyde in PBS and incubate for 20 minutes at room temperature

• Wash cells 3 times with Wash Buffer (0.1% Triton X-100 in PBS)

• Apply Quenching Buffer (Wash Buffer containing 1% H₂O₂ and 0.1% Sodium Azide) for 20 minutes to inactivate endogenous peroxidases

• Wash and block with Antibody Blocking Buffer for 1 hour prior to antibody incubation

For non-adherent cells:

• Pre-coat plates with 10 μg/mL poly-L-Lysine for 30 minutes at 37°C

• Wash twice with PBS before seeding approximately 17,000 cells per well

• Fix with 8% formaldehyde (higher concentration than for adherent cells)

• Follow the remaining washing and blocking steps as for adherent cells

This detailed protocol preserves phosphorylation status while ensuring proper cell adherence and antibody accessibility.

What positive controls should be included when working with Phospho-RELA (Ser468) Antibody?

When designing experiments with Phospho-RELA (Ser468) Antibody, include these controls:

• Stimulated positive control: Cells treated with TNF-α (20 ng/mL) and Calyculin A (50 nM) for 15 minutes induce robust phosphorylation at Ser468, serving as an excellent positive control

• Total RELA/NF-κB p65 detection: Always run parallel samples with an antibody detecting total RELA/NF-κB p65 regardless of phosphorylation status. This confirms protein presence and allows calculation of phosphorylation ratios

• Untreated negative control: Include untreated cells to establish baseline phosphorylation levels

• Phosphatase-treated control: Sample treatment with lambda phosphatase before antibody incubation confirms signal specificity to phosphorylated epitope

Including these controls enables confident interpretation of experimental results by confirming antibody functionality and signal specificity.

What treatment conditions effectively induce phosphorylation at Ser468 of RELA/NF-κB p65?

To effectively induce and study Ser468 phosphorylation of RELA/NF-κB p65, consider these validated treatment conditions:

• TNF-α treatment: Application of 20 ng/mL TNF-α for 15 minutes effectively induces Ser468 phosphorylation

• Phosphatase inhibition: Co-treatment with 50 nM Calyculin A (a serine/threonine phosphatase inhibitor) enhances and stabilizes phosphorylation events

• Serum starvation followed by stimulation: Serum starve cells for 16 hours prior to stimulation to reduce baseline phosphorylation and enhance signal-to-noise ratio after induction

When designing experiments, consider that phosphorylation dynamics are often rapid and transient, necessitating careful time-course studies to capture optimal phosphorylation windows.

How can I optimize antibody dilution and incubation conditions for maximum sensitivity?

Optimizing antibody conditions is crucial for detecting phosphorylated RELA with high sensitivity:

• Antibody dilution optimization:

  • For Western blotting: Begin with 1:1000 dilution and adjust based on signal intensity

  • For In-Cell ELISA: Dilute in Antibody Dilution Buffer with a recommended starting range of 1:100 to 1:1000

• Incubation parameters:

  • Temperature: Overnight incubation at 4°C generally yields optimal results with reduced background

  • Volume: For maximum sensitivity in ELISA formats, minimize incubation volume to as little as 25 μL (though this makes multichannel pipetting more challenging)

  • Duration: Primary antibody incubation overnight; secondary antibody for precisely 1 hour at room temperature

• Signal enhancement strategies:

  • For low abundance targets, increasing antibody concentration and reducing incubation volume can improve detection

  • For subtle phosphorylation changes in high-abundance targets, decreasing phospho-antibody concentration can enhance sensitivity to small changes

Systematic optimization through titration experiments will determine ideal conditions for your specific experimental system.

What is the proper protocol for colorimetric detection in In-Cell ELISA applications?

For reliable colorimetric detection when using Phospho-RELA (Ser468) Antibody in In-Cell ELISA:

• After secondary antibody incubation, wash cells thoroughly:

  • 3 times for 5 minutes with Wash Buffer

  • 2 times for 5 minutes with 1X PBS

• Add 100 μL Developing Solution to each well:

  • Ensure Developing Solution has equilibrated to room temperature for at least one hour before use

  • Protect from light during this equilibration

• Monitor blue color development:

  • Incubate for 2-20 minutes at room temperature protected from direct light

  • Observe until the darkest-staining wells reach medium to dark blue

  • Avoid overdevelopment which can reduce signal differentiation

• Add 100 μL Stop Solution:

  • This acidic solution converts the blue color to yellow

  • Read absorbance at 450 nm immediately after adding Stop Solution

• Normalize signal:

  • Either normalize to cell number using Crystal Violet staining

  • Or calculate the ratio of phosphorylated to total RELA/NF-κB p65 signal

This standardized protocol ensures reproducible and quantifiable results when assessing RELA phosphorylation levels.

Why might I observe weak or no signal when using Phospho-RELA (Ser468) Antibody?

When encountering weak or absent signals with Phospho-RELA (Ser468) Antibody, systematically investigate these common causes:

• Reagent issues:

  • Omission of key reagent in the protocol

  • Inactive substrate or conjugate (check expiration dates)

  • Presence of enzyme inhibitors (e.g., sodium azide in buffers)

• Technical parameters:

  • Cold Developing Solution (ensure room temperature equilibration)

  • Inadequate volume of Developing Solution

  • Excessive washing steps (limit wash steps to 5 minutes each)

• Biological factors:

  • Insufficient cell number (aim for 80% confluence)

  • Poor cell adherence to plates

  • Cells from incorrect species (confirm reactivity with your model)

  • Undetectable levels of phosphorylated protein (verify with Western blot)

  • Rapid dephosphorylation (include phosphatase inhibitors)

• Antibody factors:

  • Improper antibody dilution

  • Extended secondary antibody incubation (limit to 1 hour)

Implementation of appropriate controls, particularly the total RELA antibody which should always yield signal if the protein is present, can help diagnose the specific issue in your experimental setup.

How can I address poor precision or high background in my experiments?

To improve precision and reduce background when working with Phospho-RELA (Ser468) Antibody:

• To reduce cross-well read-through in plate assays:

  • Avoid using phospho and total antibodies in adjacent wells

  • If unavoidable, use the total antibody at a higher dilution

  • Utilize the 96-well plates designed to minimize signal cross-contamination

• To enhance signal-to-noise ratio:

  • Increase blocking time to reduce non-specific binding

  • Optimize antibody concentration (too high can increase background)

  • Ensure thorough washing between steps

  • Include negative controls (no primary antibody) to assess background levels

• To improve assay reproducibility:

  • Standardize cell density across wells

  • Maintain consistent incubation times and temperatures

  • Use calibrated pipettes for reagent addition

  • Avoid edge effects by not using outer wells or pre-incubating plates

• For difficult-to-detect phosphorylation events:

  • Pre-treat cells with phosphatase inhibitors to preserve phosphorylation

  • Optimize cell lysis conditions to maintain epitope integrity

  • Consider signal amplification systems for very low abundance targets

Methodical optimization of these parameters will significantly improve experimental precision and reliability.

How can Phospho-RELA (Ser468) Antibody be used to investigate signaling pathway crosstalk?

Investigating signaling pathway crosstalk with Phospho-RELA (Ser468) Antibody requires sophisticated experimental design:

• Multiplexed phosphorylation profiling:

  • Simultaneously assess Ser468 phosphorylation alongside other NF-κB modifications (e.g., Ser536)

  • Compare phosphorylation patterns after stimulation of different upstream pathways

  • Create a temporal map of phosphorylation events at different sites

• Inhibitor studies:

  • Apply specific kinase or phosphatase inhibitors to determine which enzymes regulate Ser468 phosphorylation

  • Assess how inhibition of other pathways (MAPK, PI3K, etc.) affects Ser468 phosphorylation status

  • Use combinatorial inhibitor treatments to uncover hierarchical relationships between pathways

• Genetic manipulation approaches:

  • Employ site-directed mutagenesis (S468A or S468D) to create phospho-null or phospho-mimetic RELA variants

  • Assess how these mutations affect responsiveness to different stimuli

  • Compare gene expression profiles downstream of wild-type versus mutant RELA

• Functional readouts:

  • Correlate Ser468 phosphorylation with transcriptional activity using reporter assays

  • Assess protein-protein interactions that may be regulated by this phosphorylation event

  • Determine nuclear/cytoplasmic distribution of RELA based on phosphorylation status

These approaches enable mechanistic insights into how multiple signaling inputs converge on RELA to regulate its function in different cellular contexts.

What are the implications of detecting differential phosphorylation patterns at Ser468 versus Ser536?

The differential phosphorylation patterns at Ser468 versus Ser536 on RELA/NF-κB p65 represent distinct regulatory mechanisms with important functional consequences:

• Functional significance:

  • Ser536 phosphorylation generally enhances transcriptional activity

  • Ser468 phosphorylation can have context-dependent effects, in some cases promoting termination of NF-κB signaling

  • The combination of these modifications creates a "phosphorylation code" that dictates RELA function

• Kinase specificity:

  • Different upstream kinases target these sites (IKKβ, GSK3β, and others for Ser468; IKKα/β for Ser536)

  • This allows for integration of multiple signaling inputs through distinct phosphorylation events

• Experimental approach for comparative analysis:

  • Use In-Cell ELISA kits containing antibodies against both phosphorylation sites to quantitatively compare their levels

  • Perform time-course experiments to determine the temporal relationship between these phosphorylation events

  • Calculate phosphorylation ratios (pSer468/pSer536) to assess the balance between potentially opposing regulatory events

• Experimental data visualization:

  Treatment	pSer468/Total RELA	pSer536/Total RELA	pSer468/pSer536 Ratio
  Untreated	Low	Low	Variable
  TNF-α	Increased	Strongly increased	Decreased
  TNF-α + Calyculin A	Strongly increased	Strongly increased	Approximately 1:1

Understanding these distinct phosphorylation events provides insights into the complex regulation of NF-κB transcriptional activity and its role in diverse biological processes.

What methodological approaches enable study of temporal dynamics of Ser468 phosphorylation?

To effectively capture the temporal dynamics of RELA Ser468 phosphorylation:

• Time-course experimental design:

  • Sample collection at multiple timepoints (0, 5, 15, 30, 60, 120, 240 minutes post-stimulation)

  • Rapid sample processing with phosphatase inhibitors to preserve phosphorylation status

  • Simultaneous assessment of multiple phosphorylation sites to establish temporal relationships

• Live-cell imaging approaches:

  • Phospho-specific fluorescent biosensors can be engineered to detect Ser468 phosphorylation in real-time

  • FRET-based reporters that undergo conformational changes upon phosphorylation

  • Correlation of phosphorylation events with subcellular localization changes

• Pulse-chase methodology:

  • Brief stimulation followed by stimulus removal and inhibitor addition

  • Tracks both phosphorylation and dephosphorylation kinetics

  • Reveals the stability and persistence of specific phosphorylation events

• Mathematical modeling integration:

  • Apply computational approaches to quantitative time-course data

  • Determine rate constants for phosphorylation/dephosphorylation reactions

  • Predict effects of perturbations on signaling dynamics

These methodological approaches reveal not just the presence or absence of phosphorylation but its dynamic regulation, providing insights into the temporal control of NF-κB signaling in various biological contexts.

How can Phospho-RELA (Ser468) Antibody be used in disease model research?

For investigating RELA Ser468 phosphorylation in disease models, implement these experimental strategies:

• Comparative analysis across disease models:

  • Compare phosphorylation levels between normal and diseased tissues/cells

  • Assess how phosphorylation patterns change during disease progression

  • Correlate phosphorylation status with disease severity markers

• Therapeutic intervention studies:

  • Measure how potential therapeutic compounds affect Ser468 phosphorylation

  • Establish temporal relationships between phosphorylation changes and therapeutic outcomes

  • Determine if Ser468 phosphorylation could serve as a biomarker for treatment response

• Genetic model integration:

  • Use CRISPR-Cas9 to create S468A (phospho-null) or S468D (phospho-mimetic) mutations

  • Assess how these mutations affect disease phenotypes

  • Compare with pharmacological interventions targeting the same pathway

• Implementation in precision medicine applications:

  • Develop standardized protocols for measuring Ser468 phosphorylation in patient samples

  • Correlate phosphorylation patterns with treatment responses

  • Establish whether Ser468 phosphorylation status could guide therapeutic decision-making

These approaches enable translation of basic phosphorylation research into potential clinical applications while maintaining scientific rigor and methodological consistency.

What considerations are important when designing ChIP experiments using Phospho-RELA (Ser468) Antibody?

When designing Chromatin Immunoprecipitation (ChIP) experiments with Phospho-RELA (Ser468) Antibody:

• Chromatin preparation optimization:

  • Use dual crosslinking (formaldehyde plus protein-specific crosslinkers) to preserve phospho-epitopes

  • Include phosphatase inhibitors throughout the protocol

  • Optimize sonication conditions to generate 200-500 bp fragments while preserving epitope integrity

• Antibody validation for ChIP applications:

  • Verify antibody specificity in ChIP conditions using phosphatase treatment controls

  • Perform sequential ChIP with total RELA antibody followed by phospho-specific antibody

  • Include IgG controls and positive control regions (known NF-κB binding sites)

• Experimental design considerations:

  • Compare binding profiles of phospho-Ser468 RELA versus total RELA

  • Perform time-course ChIP after stimulus to correlate phosphorylation with genomic binding

  • Include stimulus-specific positive controls (e.g., TNF-α-responsive genes)

• Data analysis approach:

  • Identify differential binding sites between phospho-RELA and total RELA

  • Correlate binding with gene expression changes

  • Perform motif analysis to identify co-factors that might preferentially interact with phosphorylated RELA

This methodological framework enables investigation of how Ser468 phosphorylation influences the genomic targeting and transcriptional activity of RELA in different cellular contexts.

How might single-cell analysis techniques be integrated with Phospho-RELA (Ser468) detection?

Integrating single-cell analysis with Phospho-RELA (Ser468) detection represents an emerging frontier:

• Single-cell phospho-flow cytometry:

  • Adapt Phospho-RELA (Ser468) Antibody for flow cytometry applications

  • Simultaneously measure multiple phosphorylation sites and other proteins

  • Reveal cell-to-cell variability in NF-κB phosphorylation patterns

• Mass cytometry (CyTOF) integration:

  • Develop metal-conjugated Phospho-RELA (Ser468) Antibodies

  • Simultaneously measure 30+ proteins and phosphorylation sites at single-cell resolution

  • Create high-dimensional profiles of signaling states

• Single-cell imaging advancements:

  • Apply multiplex immunofluorescence to detect multiple phosphorylation sites simultaneously

  • Correlate phosphorylation status with subcellular localization

  • Implement live-cell imaging to track phosphorylation dynamics in individual cells

• Integration with single-cell transcriptomics:

  • Combine phospho-protein detection with scRNA-seq through CITE-seq approaches

  • Correlate Ser468 phosphorylation with transcriptional profiles at single-cell resolution

  • Identify cell subpopulations with distinct signaling and gene expression patterns

These approaches will move beyond population averages to reveal how heterogeneity in NF-κB phosphorylation contributes to diverse cellular responses and phenotypes.

What standardization methods should be implemented for comparing Phospho-RELA (Ser468) data across studies?

To enhance reproducibility and comparability of Phospho-RELA (Ser468) data across research studies:

• Reference standard implementation:

  • Develop recombinant phosphorylated RELA protein standards

  • Create standardized positive control cell lysates

  • Establish common normalization methods for cross-laboratory comparison

• Protocol standardization recommendations:

  • Standardize cell fixation and permeabilization procedures

  • Establish consensus antibody dilutions and incubation parameters

  • Define common stimulation protocols (e.g., TNF-α concentration and duration)

• Reporting standards development:

  • Include detailed methods sections specifying all parameters

  • Report raw data alongside normalized values

  • Provide antibody validation data specific to each experimental system

• Calibration and quality control measures:

  • Implement regular antibody lot testing with standard samples

  • Use calibration curves with recombinant phospho-proteins

  • Include inter-assay controls to enable comparison between experiments

These standardization approaches will enhance data reproducibility and facilitate meta-analyses across multiple studies, accelerating progress in understanding the complex regulation of NF-κB signaling.