Phospho-IKBKB (Tyr199) Antibody

What is IKK-beta and why is its phosphorylation at Tyrosine 199 significant?

IKK-beta (Inhibitor of nuclear factor kappa-B kinase subunit beta) is a key serine/threonine protein kinase essential in the NF-kappa-B signaling pathway. This pathway is activated by multiple stimuli including inflammatory cytokines, bacterial or viral products, DNA damage, and other cellular stresses .

Phosphorylation at Tyrosine 199 represents a critical post-translational modification that modulates IKK-beta's activity and function. While serine phosphorylation sites (particularly Ser-177 and Ser-181) have been well-characterized in activating IKK-beta, tyrosine phosphorylation at the 199 position offers an additional regulatory mechanism that affects pathway dynamics and potentially cross-talk with other signaling cascades .

The significance of this specific modification lies in its ability to fine-tune inflammatory responses. Dysregulation of IKK-beta phosphorylation, including at Tyr199, has been implicated in various pathological conditions such as chronic inflammation, cancer, and autoimmune disorders .

How does Tyr199 phosphorylation differ from other post-translational modifications of IKK-beta?

IKK-beta undergoes multiple post-translational modifications that regulate its function:

Tyr199 phosphorylation appears to play a unique role compared to the well-characterized serine phosphorylation sites, potentially mediating cross-talk between tyrosine kinase pathways and the canonical NF-kappa-B signaling pathway .

What cellular conditions induce IKK-beta Tyr199 phosphorylation?

Phosphorylation of IKK-beta at Tyr199 can be induced by several cellular conditions and stimuli:

• Inflammatory cytokines (TNF-α, IL-1β)

• Bacterial or viral components (LPS, viral proteins)

• Cellular stress conditions

• Growth factor receptor activation

• Reactive oxygen species

These stimuli activate specific tyrosine kinases that target IKK-beta at the Tyr199 position. Importantly, the phosphorylation state at this residue can be dynamically regulated through the coordinated action of both kinases and phosphatases, allowing for temporal control of IKK-beta activity in response to changing cellular environments .

What are the recommended applications for Phospho-IKBKB (Tyr199) antibodies?

Based on the technical data from multiple manufacturers, Phospho-IKBKB (Tyr199) antibodies can be utilized in several experimental applications:

The antibody detects endogenous levels of IKK-beta protein only when phosphorylated at Tyr199, providing specificity for studying this particular modification .

How should samples be prepared for optimal detection of Phospho-IKBKB (Tyr199)?

For optimal detection of Phospho-IKBKB (Tyr199), consider these preparation methods based on application:

For Western Blot:

• Rapidly harvest cells/tissues in the presence of phosphatase inhibitors

• Lyse samples in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease/phosphatase inhibitor cocktails

• Clear lysates by centrifugation (14,000 × g, 15 min, 4°C)

• Determine protein concentration and load 20-50 μg per lane

• Separate proteins using 8-10% SDS-PAGE gels

For Immunofluorescence:

• Culture cells on coverslips or chamber slides

• Fix with methanol (−20°C, 10 minutes) for optimal epitope preservation

• Include phosphatase inhibitors in all buffers

• Block with 5% BSA in TBST to reduce background

For Immunohistochemistry:

• Fix tissues with 4% paraformaldehyde

• Use heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Block endogenous peroxidase activity with hydrogen peroxide

• Use PBS with phosphatase inhibitors for all washing steps

What controls should be included when using Phospho-IKBKB (Tyr199) antibodies?

Rigorous experimental design requires appropriate controls:

Positive Controls:

• Cells treated with cytokines (TNF-α, IL-1β) to induce IKK-beta phosphorylation

• Cell lines with constitutively active IKK-beta signaling (e.g., certain cancer cell lines)

• Recombinant phosphorylated IKK-beta protein (for Western blot)

Negative Controls:

• Untreated cells with minimal basal phosphorylation

• Cells pre-treated with phosphatase before antibody incubation

• Immunizing phosphopeptide competition assay

• Blocking peptide corresponding to the phospho-epitope

• Secondary antibody-only control

Technical Controls:

• Non-phospho-specific IKK-beta antibody to verify total protein expression

• Housekeeping protein detection (for Western blot loading control)

• Phospho-null mutant (Y199F) transfected cells

Including these controls ensures the specificity of the signal detected and helps troubleshoot any issues with the experimental setup .

Why might I be getting weak or no signal when using Phospho-IKBKB (Tyr199) antibodies?

Several factors can contribute to weak or absent signals:

• Low phosphorylation levels:

  • Phosphorylation may be transient or present at low levels

  • Solution: Optimize stimulation conditions; use phosphatase inhibitors immediately upon cell lysis

• Protein degradation:

  • Rapid dephosphorylation during sample preparation

  • Solution: Keep samples cold; use fresh phosphatase inhibitor cocktails; process samples quickly

• Antibody-related issues:

  • Improper storage or handling affecting antibody activity

  • Solution: Avoid repeated freeze-thaw cycles; store at recommended temperature (-20°C)

• Technical parameters:

  • Suboptimal antibody dilution or incubation conditions

  • Solution: Titrate antibody concentration; extend incubation time; optimize blocking conditions

• Sample preparation:

  • Loss of phospho-epitope during fixation (for IHC/IF)

  • Solution: Test different fixation methods; ensure proper epitope retrieval procedures

If standard troubleshooting doesn't resolve the issue, consider phospho-enrichment techniques such as immunoprecipitation with total IKK-beta antibody followed by phospho-detection .

How can I distinguish between phosphorylated and non-phosphorylated forms of IKBKB?

Distinguishing between phosphorylated and non-phosphorylated IKBKB requires strategic approaches:

• Parallel antibody usage:

  • Use both phospho-specific and total IKK-beta antibodies on parallel samples

  • Calculate the ratio of phosphorylated to total protein

• Phosphatase treatment:

  • Split your sample and treat one portion with lambda phosphatase

  • Compare treated vs. untreated samples to confirm phospho-specificity

• 2D gel electrophoresis:

  • Separate proteins based on isoelectric point and molecular weight

  • Phosphorylated forms typically show acidic shifts

• Phos-tag™ SDS-PAGE:

  • Use Phos-tag™ acrylamide gels to retard migration of phosphorylated proteins

  • Creates distinct bands for phosphorylated and non-phosphorylated forms

• Mass spectrometry:

  • Analyze tryptic digests to identify and quantify phosphorylated peptides

  • Provides site-specific information on phosphorylation status

How can Phospho-IKBKB (Tyr199) antibodies be used to study the NF-kappa-B pathway in inflammatory diseases?

Phospho-IKBKB (Tyr199) antibodies offer powerful tools for investigating NF-kappa-B dysregulation in inflammatory conditions:

• Disease model characterization:

  • Profile Tyr199 phosphorylation status in tissue samples from inflammatory disease models

  • Compare with healthy controls to identify disease-specific alterations

• Drug discovery applications:

  • Screen compounds for their ability to modulate IKK-beta Tyr199 phosphorylation

  • Evaluate effects on downstream NF-kappa-B target gene expression

• Pathway cross-talk analysis:

  • Investigate interactions between IKK-beta and other signaling pathways

  • Determine how Tyr199 phosphorylation influences these interactions

• Time-course studies:

  • Monitor dynamic changes in IKK-beta phosphorylation following inflammatory stimuli

  • Correlate with disease progression or therapeutic response

• Cellular localization:

  • Examine subcellular distribution of phosphorylated IKK-beta using immunofluorescence

  • Determine how localization correlates with NF-kappa-B activation states

What techniques can be combined with Phospho-IKBKB (Tyr199) antibodies for comprehensive phosphorylation analysis?

Advanced research often requires integrating multiple techniques:

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions involving phosphorylated IKK-beta

  • Visualize complexes with spatial resolution in fixed cells

• ChIP-sequencing following IKK-beta immunoprecipitation:

  • Identify genomic targets regulated by phosphorylated IKK-beta

  • Map the nuclear functions of phosphorylated IKK-beta

• Phosphoproteomics:

  • Combine phospho-IKBKB antibodies with mass spectrometry

  • Identify novel phosphorylation sites and quantify relative abundances

• Multiplex immunoassays:

  • Simultaneously measure multiple phosphorylated proteins in the NF-kappa-B pathway

  • Analyze pathway activation patterns in complex samples

• Live-cell imaging with phospho-biosensors:

  • Develop FRET-based reporters for real-time monitoring of IKK-beta phosphorylation

  • Track spatiotemporal dynamics of phosphorylation events

How should I design experiments to study the dynamics of IKBKB Tyr199 phosphorylation?

Effective experimental design for studying IKBKB Tyr199 phosphorylation dynamics:

• Stimulation optimization:

  • Determine appropriate stimulus concentration and duration

  • Include multiple time points (0, 5, 15, 30, 60, 120 minutes) to capture phosphorylation kinetics

  • Consider using reversible inhibitors to study dephosphorylation rates

• Genetic approaches:

  • Use CRISPR/Cas9 to generate Y199F mutants for functional studies

  • Create phosphomimetic mutants (Y199E/D) to simulate constitutive phosphorylation

  • Compare phenotypes of wildtype vs. mutant cells

• Inhibitor studies:

  • Test kinase inhibitors to identify enzymes responsible for Tyr199 phosphorylation

  • Use phosphatase inhibitors to stabilize phosphorylation status

  • Implement dose-response designs to determine IC50 values

• Single-cell analysis:

  • Apply flow cytometry or mass cytometry with phospho-specific antibodies

  • Identify heterogeneity in phosphorylation responses within cell populations

  • Correlate with other cellular parameters

• In vivo models:

  • Design tissue collection protocols that preserve phosphorylation status

  • Consider using in vivo phosphorylation bioassays

  • Plan for adequate biological replicates (minimum n=5 per condition)

What cell stimulation protocols are optimal for inducing IKBKB Tyr199 phosphorylation?

Based on the current understanding of IKK-beta regulation, these stimulation protocols can effectively induce Tyr199 phosphorylation:

For all stimulation protocols:

• Serum-starve cells for 4-6 hours before stimulation

• Perform stimulation at 37°C, 5% CO₂

• Terminate stimulation by rapid media removal and cell lysis in cold buffer containing phosphatase inhibitors

• Include unstimulated controls and multiple time points to capture phosphorylation dynamics

How can I quantify changes in IKBKB Tyr199 phosphorylation levels?

Accurate quantification of phosphorylation changes is essential for rigorous research:

• Western blot densitometry:

  • Normalize phospho-IKK-beta signal to total IKK-beta

  • Use digital image analysis software with linear dynamic range

  • Include standard curves with recombinant phosphoprotein

• ELISA-based quantification:

  • Utilize cell-based ELISA kits specifically designed for Phospho-IKBKB (Tyr199)

  • Calculate phosphorylation index as ratio of phospho-signal to total protein

  • Construct standard curves for absolute quantification

• Phospho-flow cytometry:

  • Analyze phosphorylation at single-cell resolution

  • Measure mean fluorescence intensity (MFI) of phospho-signal

  • Present data as fold-change in MFI or percentage of positive cells

• Mass spectrometry:

  • Use SILAC or TMT labeling for relative quantification

  • Calculate phosphopeptide abundance ratios between conditions

  • Implement parallel reaction monitoring for targeted quantification

• Automated high-content imaging:

  • Measure subcellular distribution and intensity of phospho-signals

  • Analyze hundreds of cells per condition for statistical robustness

  • Quantify nuclear/cytoplasmic ratios to infer functional consequences