Phospho-IKBKB (Tyr188) Antibody

What is IKBKB and why is tyrosine 188 phosphorylation significant?

IKBKB (Inhibitor of nuclear factor kappa-B kinase subunit beta, also known as IKK-beta) is a serine kinase that plays an essential role in the NF-kappa-B signaling pathway. This pathway is activated by multiple stimuli including inflammatory cytokines, bacterial or viral products, DNA damage, and cellular stress . IKBKB acts within the canonical IKK complex to phosphorylate inhibitors of NF-kappa-B on critical serine residues, leading to their ubiquitination and degradation, which allows free NF-kappa-B to translocate to the nucleus .

While IKBKB primarily functions as a serine/threonine kinase, its own activity is regulated through various post-translational modifications, including phosphorylation at tyrosine 188. This specific phosphorylation site affects IKBKB function and signaling dynamics. Phospho-specific antibodies that exclusively recognize IKBKB when phosphorylated at Tyr188 enable researchers to investigate this regulatory mechanism across experimental conditions .

How does the Phospho-IKBKB (Tyr188) Antibody differ from other IKBKB antibodies?

Phospho-IKBKB (Tyr188) Antibody specifically detects endogenous levels of IKBKB only when phosphorylated at tyrosine 188, unlike general IKBKB antibodies that detect total protein regardless of phosphorylation status . These antibodies are typically generated by immunizing animals with synthetic phosphopeptides corresponding to the region surrounding Tyr188 (often amino acids 130-210 in human IKBKB) .

The specificity is achieved through a rigorous purification process that typically involves:

• Affinity purification from antiserum using phosphopeptide chromatography

• Removal of non-phospho-specific antibodies through non-phosphopeptide chromatography

• Validation through blocking experiments with phospho-peptides

This specificity allows researchers to monitor the phosphorylation state at this particular residue without interference from unphosphorylated IKBKB or other phosphorylation sites, enabling precise study of this regulatory mechanism in various signaling contexts.

What are the principal applications for Phospho-IKBKB (Tyr188) Antibody in current research?

The Phospho-IKBKB (Tyr188) Antibody has multiple research applications:

Western Blotting (WB): The most common application, with recommended dilution ranges of 1:500-1:2000 . This technique allows for quantitative assessment of phosphorylation levels in cell and tissue lysates.

Immunohistochemistry (IHC-P): Used to visualize the spatial distribution of phosphorylated IKBKB within tissue sections, typically at dilutions of 1:100-1:300 . This is particularly valuable in cancer research, as demonstrated in studies using human breast carcinoma samples .

Immunofluorescence (IF): Enables subcellular localization studies of phosphorylated IKBKB, particularly useful for examining nuclear translocation and cytoplasmic distribution patterns.

ELISA: Allows quantitative measurement at higher dilutions (1:10000) , enabling high-throughput screening approaches.

The antibody shows reactivity across multiple species including human, mouse, rat, and monkey samples, making it versatile for comparative studies across model organisms .

What controls are essential when validating Phospho-IKBKB (Tyr188) Antibody specificity?

Rigorous validation of phospho-specific antibodies requires a multi-faceted approach:

• Peptide Competition Assays: Pre-incubation with the phosphopeptide immunogen should abolish specific signal. Several commercial products include this validation, demonstrating signal elimination when the antibody is blocked with the phospho-peptide .

• Phosphatase Treatment Controls: Samples treated with phosphatases should show reduced or eliminated signal compared to untreated samples, confirming phosphorylation-dependent recognition.

• Stimulation/Inhibition Experiments:

  • Positive controls: Samples treated with agents known to increase Tyr188 phosphorylation

  • Negative controls: Samples treated with IKBKB inhibitors or kinase-dead mutants

• Genetic Controls: Ideally, IKBKB knockout/knockdown samples should be used to confirm antibody specificity and eliminate potential cross-reactivity.

• Cross-validation: Comparing results across different techniques (WB, IHC, IF) and with different antibody clones targeting the same phosphorylation site provides additional confidence in specificity.

Data from validation experiments should be carefully documented and included in publications to support the reliability of findings.

How should samples be prepared to preserve IKBKB phosphorylation status?

Preserving phosphorylation status requires careful attention to multiple factors:

Tissue/Cell Collection:

• Rapidly harvest and process samples to minimize post-collection changes

• Flash-freeze tissues in liquid nitrogen immediately after collection

• For cell culture, avoid prolonged washing steps that might activate phosphatases

Lysis Buffer Composition:

• Include comprehensive phosphatase inhibitor cocktails targeting both serine/threonine and tyrosine phosphatases

• Use fresh inhibitors prepared immediately before extraction

• Include appropriate detergents (e.g., NP-40, Triton X-100) for efficient protein extraction

• Maintain cold temperature (4°C) throughout processing

Storage Conditions:

• Store samples at -20°C (short-term) or -80°C (long-term)

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Include 50% glycerol for stabilization during storage

Antigen Retrieval (for IHC):

• For paraffin-embedded tissues, use high-pressure and high-temperature antigen retrieval with Tris-EDTA buffer (pH 8.0)

• Optimize retrieval conditions specifically for phospho-epitopes, which may be more sensitive than total protein detection

Following these precautions is critical as phosphorylation marks, particularly on tyrosine residues, can be rapidly lost during sample preparation.

How can inconsistent Western blot results with Phospho-IKBKB (Tyr188) Antibody be resolved?

Inconsistent Western blot results with phospho-specific antibodies are common technical challenges that can be systematically addressed:

Sample-Related Factors:

• Ensure consistent protein extraction efficiency across samples

• Verify equal protein loading through total protein stains (Ponceau S) and loading controls

• Consider that phosphorylation states can change rapidly during manipulation

Technical Optimization:

• Test multiple antibody concentrations (1:500-1:2000 range recommended)

• Optimize blocking conditions to reduce background (typically BSA-based blockers work better than milk for phospho-epitopes)

• Adjust transfer conditions for IKBKB's high molecular weight (~87 kDa)

• Consider using PVDF membranes instead of nitrocellulose for better protein retention

Antibody-Specific Considerations:

• Store antibody according to manufacturer recommendations (-20°C)

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Consider the formulation buffer (typically PBS with 50% glycerol and 0.5% BSA)

• Test different antibody lots if available

Biological Variability:

• Account for dynamic phosphorylation events that may vary with cell culture conditions

• Consider that signaling pathways often show temporal fluctuations

• Standardize treatment conditions and timing precisely

A systematic troubleshooting approach testing each variable independently will help identify the source of inconsistency.

What optimization strategies are recommended for immunohistochemistry with Phospho-IKBKB (Tyr188) Antibody?

Successful immunohistochemistry with phospho-specific antibodies requires specialized optimization:

Tissue Preparation:

• Fixation method and duration significantly impact phospho-epitope preservation

• Phosphorylation marks are often better preserved in frozen tissues than in FFPE samples

• If using FFPE tissues, minimize fixation time while ensuring adequate tissue morphology

Antigen Retrieval:

• High-pressure and high-temperature conditions with Tris-EDTA buffer (pH 8.0) are recommended

• Optimize time and temperature empirically for your specific tissue type

• Use consistent retrieval conditions for all experimental samples

Antibody Optimization:

• Test a range of antibody dilutions (1:100-1:300 recommended for IHC)

• Extend primary antibody incubation time (overnight at 4°C often yields best results)

• Use detection systems with appropriate sensitivity (HRP-polymer systems often provide better signal-to-noise ratio than ABC methods)

Controls:

• Always run parallel sections with phospho-peptide blocking to confirm signal specificity

• Include positive control tissues known to express phosphorylated IKBKB

• Include technical negative controls (primary antibody omission, isotype controls)

Signal Interpretation:

• Establish clear scoring criteria for phospho-IKBKB positivity

• Consider both intensity and distribution patterns

• Use digital image analysis when possible for quantitative assessment

Successful phospho-protein IHC often requires more extensive optimization than standard IHC protocols.

How should researchers quantify and normalize phospho-IKBKB (Tyr188) levels in experimental samples?

Accurate quantification of phosphorylation status requires careful normalization strategies:

Western Blot Quantification:

• Use digital image analysis software (ImageJ, Image Lab, etc.) for densitometry

• Apply appropriate normalization strategies:

  • Primary approach: Normalize phospho-IKBKB to total IKBKB levels

  • Secondary approach: Include housekeeping protein controls (β-actin, GAPDH)

• Run standard curves with varying protein amounts to ensure measurements fall within the linear range

• Perform statistical analysis across multiple biological replicates (n≥3)

ELISA-Based Quantification:

• Develop standard curves using recombinant phosphorylated and non-phosphorylated IKBKB

• Express results as the ratio of phosphorylated to total protein

• Include inter-plate calibrators for multi-plate experiments

Flow Cytometry Quantification:

• Use median fluorescence intensity (MFI) rather than mean values

• Compare phospho-signal to isotype controls

• Include unstimulated controls as baseline references

Immunohistochemistry Quantification:

• Use digital pathology tools to quantify staining intensity

• Develop scoring systems incorporating both intensity and percentage of positive cells

• Consider spatial heterogeneity in phosphorylation patterns

Regardless of method, always report both raw data and normalized values with appropriate statistical analysis.

How do researchers interpret contradictory findings regarding IKBKB phosphorylation across different experimental systems?

Contradictory findings in phosphorylation studies often arise from methodological differences and context-dependent signaling. A systematic approach to reconciling disparate results includes:

Methodological Considerations:

• Different antibodies may recognize distinct conformational states or epitopes even when targeting the same phosphorylation site

• Sensitivity thresholds vary between techniques (mass spectrometry vs. immunoblotting)

• Sample preparation methods affect phosphorylation preservation

Biological Context Variations:

• Cell type-specific signaling networks may produce different phosphorylation patterns

• Temporal dynamics of phosphorylation events may lead to differences if samples are collected at different timepoints

• Microenvironmental factors (growth factors, cell density, oxygen levels) influence phosphorylation status

Reconciliation Strategies:

• Direct comparison using standardized protocols across different experimental systems

• Use of multiple detection methods within the same experimental setup

• Detailed characterization of phosphorylation kinetics rather than single timepoint measurements

• Integration of computational modeling approaches to predict context-dependent outcomes

The search results highlight a relevant example: "Paradoxically, other studies showed that pharmacological inhibition of IKBKB leads to the same results in cells" , emphasizing that contradictory findings often reflect complex regulatory mechanisms rather than experimental errors.

What is the relationship between IKBKB Tyr188 phosphorylation and its known serine phosphorylation sites?

IKBKB regulation involves complex interplay between multiple phosphorylation sites:

Key Phosphorylation Sites:

• Tyr188: The focus of specific antibodies discussed here

• Ser733: Another crucial regulatory site with dedicated antibodies available

• Other serine residues involved in activation and regulation

Regulatory Relationships:

• Sequential Phosphorylation: Phosphorylation at certain sites may be prerequisites for modification at other sites

• Crosstalk: Tyrosine phosphorylation may influence accessibility or susceptibility of serine sites to kinases/phosphatases

• Competitive Effects: Different phosphorylation events may compete for overlapping binding partners

Functional Consequences:

• Different phosphorylation patterns likely direct IKBKB toward specific substrates

• The combination of phosphorylation marks determines subcellular localization

• Temporal sequence of phosphorylation events impacts duration of signaling

Research Approaches:

• Mass spectrometry-based phosphoproteomics can identify multiple simultaneous modifications

• Site-directed mutagenesis studies (Y188F, S733A) help disentangle individual contributions

• Time-course studies with phospho-specific antibodies reveal sequential relationships

Understanding this multi-site phosphorylation code is critical for developing targeted therapeutic approaches that modulate specific IKBKB functions.

How does IKBKB Tyr188 phosphorylation contribute to disease pathogenesis based on current research?

Research has implicated IKBKB phosphorylation in several disease contexts:

Immune Disorders:
Recent studies have identified a novel germline missense mutation in human IKBKB that confers gain of function and results in a combined immune deficiency syndrome . This mutation leads to:

• Increased and prolonged IκBα phosphorylation

• Enhanced phospho-p65 levels, particularly in T cells

• Altered immune cell function, leading to clinical immunodeficiency

Neurodegenerative Diseases:
IKBKB has been shown to reduce huntingtin aggregation by phosphorylating serine 13 on huntingtin protein . This research demonstrated:

• IKBKB can regulate endogenous S13 huntingtin phosphorylation

• This regulation depends on IKBKB's kinase activity

• The effect is shared with IKBKE but not with IKBKA

• Monomeric and NEMO binding-incompetent IKBKB remain capable of increasing pS13 HTT levels

Cancer Biology:
Phosphorylated IKBKB has been detected in various cancer types, including:

• Human breast carcinoma, as demonstrated by immunohistochemistry analysis

• Potential roles in promoting cancer cell survival and proliferation through NF-κB activation

Understanding the specific role of Tyr188 phosphorylation in these disease contexts requires further investigation, but phospho-specific antibodies provide valuable tools for such studies.

What novel technical approaches are being developed to study IKBKB phosphorylation dynamics?

Several cutting-edge approaches are enhancing our ability to study phosphorylation dynamics:

Ultrasensitive Detection Methods:
Recent research has developed "novel ultrasensitive assays for measuring endogenous levels of S13 HTT phosphorylation" in the context of IKBKB research . Similar approaches could be applied to studying Tyr188 phosphorylation with enhanced sensitivity.

Live-Cell Imaging:

• FRET-based biosensors that report on IKBKB phosphorylation status in real-time

• Phospho-specific nanobodies combined with fluorescent tags for live visualization

• Optogenetic approaches to control kinase activity with spatiotemporal precision

Single-Cell Phosphoproteomics:

• Mass cytometry (CyTOF) with phospho-specific antibodies

• Single-cell Western blotting technologies

• Microfluidic platforms for analyzing phosphorylation in rare cell populations

CRISPR-Based Approaches:

• Generation of endogenously tagged IKBKB for monitoring physiological dynamics

• Knock-in mutations that mimic or prevent specific phosphorylation events

• CRISPR-activation/interference systems to modulate kinases/phosphatases affecting IKBKB

Computational Modeling:

• Integration of phosphorylation data into signaling network models

• Prediction of context-dependent outcomes of IKBKB phosphorylation

• Machine learning approaches to identify patterns in complex phosphorylation datasets

These emerging technologies promise to provide unprecedented insights into the dynamic regulation of IKBKB phosphorylation in various biological contexts.

How can researchers distinguish between direct and indirect effects on IKBKB Tyr188 phosphorylation in complex signaling networks?

Distinguishing direct versus indirect effects on phosphorylation requires sophisticated experimental approaches:

In Vitro Kinase Assays:

• Recombinant kinase and substrate proteins in cell-free systems

• ATP-dependent phosphorylation monitored by mass spectrometry

• Competitive inhibition studies to identify direct kinase-substrate relationships

Phosphatase Studies:

• Identification of phosphatases acting directly on Tyr188

• Use of phosphatase inhibitors with varying specificity profiles

• In vitro dephosphorylation assays with purified components

Kinetic Analysis:

• High-resolution time-course experiments to identify temporal order of events

• Mathematical modeling of phosphorylation/dephosphorylation rates

• Comparison of phosphorylation kinetics across multiple pathway components

Proximity-Based Approaches:

• Proximity ligation assays (PLA) to detect direct interactions between IKBKB and potential kinases/phosphatases

• BioID or APEX2 proximity labeling to identify proteins in close proximity to IKBKB

• FRET/BRET-based sensors to detect direct protein-protein interactions

Genetic Approaches:

• Specific mutagenesis of potential upstream kinases/phosphatases

• Orthogonal expression systems to introduce components sequentially

• Genetic epistasis experiments to establish pathway order

These approaches can help construct accurate signaling maps and identify direct regulatory mechanisms affecting IKBKB Tyr188 phosphorylation.

What is the significance of phosphatases in regulating IKBKB phosphorylation status?

Phosphatases play a crucial role in IKBKB regulation that is often underappreciated:

Identified Phosphatase Relationships:
Research has highlighted roles for specific phosphatases: "We sought to investigate this discrepancy by interrogating the role of phosphatases in regulating the effects of IKBKB on pS13 HTT levels. Phosphatases may affect pS13 HTT levels in different ways, including direct dephosphorylation of pS13 and regulation of the catalytic activity, localization, and stability of S13 kinases and phosphatases" .

Regulatory Mechanisms:

• Direct Dephosphorylation: Phosphatases directly removing phosphate groups from Tyr188

• Indirect Regulation: Phosphatases affecting upstream components that regulate IKBKB phosphorylation

• Feedback Control: Phosphatase activity itself regulated by IKBKB-dependent pathways

• Spatial Regulation: Phosphatases controlling subcellular localization of IKBKB

Experimental Approaches:

• Phosphatase inhibitor studies with varying specificity profiles

• siRNA/shRNA screens targeting phosphatase catalytic and regulatory subunits

• Phosphatase substrate trapping mutants to identify direct interactions

• Mass spectrometry phosphoproteomics following phosphatase manipulation

Therapeutic Implications:

• Phosphatase modulators may provide alternative approaches to kinase inhibitors

• Combination therapies targeting both kinases and phosphatases might achieve more precise pathway modulation

• Phosphatase activity biomarkers could help predict treatment responses

Understanding the dynamic interplay between kinases and phosphatases is essential for comprehending IKBKB regulation in health and disease.