Phospho-NFKBIB (Thr19) Antibody

What is the functional significance of Thr19 phosphorylation in NFKBIB compared to other phosphorylation sites?

The phosphorylation of NFKBIB (also known as IκB-β) at Thr19 represents a critical regulatory mechanism in the NF-κB signaling pathway. While Ser23 is often considered the primary phosphorylation site, Thr19 has been identified as an important phosphorylation site specifically in human IκB-β. Phosphorylation at this position occurs alongside Ser23 and contributes to the regulation of IκB-β stability and function. Unlike the rapid phosphorylation and degradation seen with IκBα, the phosphorylation and ubiquitin-mediated degradation of IκB-β occurs with much slower kinetics, creating a distinct temporal profile for NF-κB activation . This differential regulation allows for a more sustained activation of NF-κB target genes involved in inflammation, immunity, cell proliferation, and apoptosis responses. Research demonstrates that mutation of Thr19 can alter the degradation kinetics of IκB-β, highlighting its importance in regulating NF-κB activity in response to specific stimuli such as TNF-α.

How does NFKBIB (IκB-β) differ functionally from other IκB family members in the NF-κB pathway?

NFKBIB represents a distinct member of the IκB family with unique functional characteristics that differentiate it from other members such as IκBα and IκBε. NFKBIB is a 356-amino acid protein that inhibits NF-κB by forming complexes that trap it in the cytoplasm . Unlike IκBα, which undergoes rapid phosphorylation and degradation, NFKBIB exhibits significantly slower phosphorylation and ubiquitin-mediated degradation kinetics . This difference creates a more sustained activation profile for NF-κB-dependent gene expression. The human sequence of IκB-β contains a threonine at position 19, whereas other species or IκB family members may have different residues at equivalent positions . This sequence variation contributes to species-specific regulation of the NF-κB pathway and explains why some antibodies show specific reactivity only to human samples. Additionally, NFKBIB can be found in both nuclear and cytoplasmic compartments, suggesting additional regulatory functions beyond cytoplasmic retention of NF-κB .

What are the upstream kinases responsible for Thr19 phosphorylation in NFKBIB?

The primary kinases responsible for phosphorylation of NFKBIB at Thr19 are members of the IκB kinase (IKK) complex. This complex typically consists of catalytic subunits (IKKα and IKKβ) and a regulatory subunit (IKKγ/NEMO). Research indicates that IKK-mediated phosphorylation of IκB-β occurs primarily at Thr19 and Ser23 in human cells . Various stimuli, including inflammatory cytokines like TNF-α, can activate the IKK complex, leading to NFKBIB phosphorylation . Western blot analysis has shown increased phosphorylation at Thr19 in 293 cells following TNF-α treatment (20ng/ml for 30 minutes) . The specificity of these kinase activities can be verified using kinase inhibitors or through in vitro kinase assays with recombinant IKK components. Understanding the kinases involved provides important insights into the regulation of NF-κB signaling and potential therapeutic targets for modulating inflammatory responses in various disease contexts.

What are the optimal sample preparation methods for detecting phospho-NFKBIB (Thr19) in Western blot applications?

For optimal detection of phospho-NFKBIB (Thr19) in Western blot applications, sample preparation requires careful attention to preserve phosphorylation status. Cells should be lysed in buffer containing phosphatase inhibitors (such as sodium fluoride, sodium orthovanadate, and β-glycerophosphate) to prevent dephosphorylation during sample processing. For cell stimulation experiments, treating cells with TNF-α (20ng/ml for 30 minutes) has been shown to effectively induce Thr19 phosphorylation . When preparing protein samples, maintain cold conditions throughout and include protease inhibitors to prevent protein degradation.

The recommended protein loading amount is typically 30-50 μg per lane, with separation on 10-12% SDS-PAGE gels. After transfer to PVDF or nitrocellulose membranes, blocking should be performed with 5% BSA (not milk, which contains phosphatases) in TBST. For antibody incubation, a dilution range of 1:500-1:2000 is typically effective for phospho-NFKBIB (Thr19) antibodies . Including both phospho-specific and total NFKBIB antibody controls in parallel samples is essential for result interpretation. Additionally, a phospho-peptide blocking control can verify antibody specificity, as demonstrated in validation images showing signal reduction when the antibody is pre-absorbed with the immunogen peptide .

How can researchers optimize immunohistochemistry protocols for phospho-NFKBIB (Thr19) antibodies in different tissue types?

Optimizing immunohistochemistry (IHC) protocols for phospho-NFKBIB (Thr19) antibodies requires tissue-specific considerations. For formalin-fixed paraffin-embedded (FFPE) tissues, effective antigen retrieval is crucial due to phospho-epitope masking during fixation. High-pressure and high-temperature antigen retrieval using Tris-EDTA buffer (pH 8.0) has been validated for human brain tissue samples . The antibody dilution typically ranges from 1:100-1:300 for IHC applications, with overnight incubation at 4°C providing optimal results .

For frozen sections, fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.3% Triton X-100 is recommended. When working with neural tissues, which show constitutive NF-κB activity, additional optimization steps may be necessary. Background reduction can be achieved using hydrogen peroxide blocking (3% H₂O₂ for 10 minutes) before antibody incubation. Validation of specificity should include negative controls where the primary antibody is pre-absorbed with the immunogenic phospho-peptide, which should eliminate specific staining . For multiple tissue analysis, researchers should optimize the protocol for each tissue type individually, as fixation requirements and background issues can vary significantly between tissues like brain, liver, and immune tissues where NF-κB signaling is prominent.

What are the recommended protocols for quantifying phospho-NFKBIB (Thr19) levels using ELISA techniques?

For quantitative assessment of phospho-NFKBIB (Thr19) levels using ELISA techniques, both commercially available kits and custom protocols can be employed. When using commercial phospho-NFKBIB ELISA kits, follow manufacturer protocols with sample dilutions typically starting at 1:5000 . For cell-based ELISA approaches, the BosterBio IkappaB-beta (Phospho-Thr19) Colorimetric Cell-Based ELISA Kit recommends plating >5000 cells per well for optimal detection sensitivity .

For developing custom ELISA protocols, coat high-binding 96-well plates with capture antibody against total NFKBIB overnight at 4°C. After blocking with 5% BSA in PBST, add cell or tissue lysates prepared with phosphatase inhibitors. For detection, use phospho-specific antibodies at optimized dilutions (typically 1:5000 for ELISA applications) , followed by HRP-conjugated secondary antibodies. Quantification should include a standard curve using recombinant phosphorylated protein or phosphopeptide standards. The signal-to-noise ratio can be improved by including additional washing steps with PBST. For comparative studies, it's essential to normalize phospho-NFKBIB levels to total NFKBIB protein by running parallel ELISAs with phospho-specific and total protein antibodies on the same samples. This approach provides a phosphorylation index that accounts for variations in total protein expression across samples.

How can researchers distinguish between Thr19 and Ser23 phosphorylation events in the regulation of NFKBIB function?

Distinguishing between Thr19 and Ser23 phosphorylation events requires strategic experimental approaches that can isolate the individual contributions of each site. Researchers should employ site-specific phospho-antibodies that exclusively recognize either phospho-Thr19 or phospho-Ser23, such as the antibodies described in the search results . To validate specificity, western blot experiments with phospho-peptide competition can confirm antibody selectivity, as demonstrated in validation images where antibody signal is blocked by pre-absorption with the immunogen peptide .

For functional studies, site-directed mutagenesis creating T19A and S23A single mutants and T19A/S23A double mutants allows for dissection of the individual contributions of each phosphorylation site. These mutants can be expressed in cellular systems to examine effects on NF-κB activation kinetics, protein stability, and target gene expression. Mass spectrometry approaches provide another valuable method, where phosphorylated NFKBIB is immunoprecipitated and analyzed by LC-MS/MS to quantify the relative abundance of each phosphorylation site under different conditions. Temporal analysis is particularly important, as evidence suggests these phosphorylation events may occur sequentially rather than simultaneously. Kinetic studies using pulse-chase experiments with these mutants can reveal how each phosphorylation site contributes to the degradation timeline of NFKBIB following stimulation with TNF-α or other NF-κB activators.

What are the experimental approaches to study the temporal dynamics of NFKBIB (Thr19) phosphorylation in inflammation models?

Studying the temporal dynamics of NFKBIB (Thr19) phosphorylation in inflammation models requires time-course experiments with precise sample collection. Researchers should design experiments with multiple timepoints (0, 5, 15, 30, 60, 120 minutes, and extending to 24 hours) following stimulation with inflammatory inducers such as TNF-α (20ng/ml), IL-1β, or LPS. Western blot analysis using phospho-specific antibodies can track the phosphorylation status at each timepoint, as demonstrated in validation studies where TNF-α treatment (20ng/ml for 30 minutes) induced detectable phosphorylation .

For in vivo inflammation models, tissue samples should be collected at defined intervals after inflammatory challenge and immediately preserved to maintain phosphorylation status. Immunohistochemistry with phospho-NFKBIB (Thr19) antibodies can reveal the spatial and temporal distribution of phosphorylation events in tissue contexts . Live-cell imaging approaches using fluorescent reporters coupled to phospho-binding domains can provide real-time visualization of phosphorylation dynamics in cellular models. Computational modeling can integrate these temporal datasets to predict the kinetic parameters governing NFKBIB phosphorylation and subsequent NF-κB activation. This approach is particularly valuable given the evidence that IκB-β phosphorylation and degradation occur with slower kinetics compared to IκBα , creating distinct temporal profiles for NF-κB activation that influence downstream gene expression patterns in inflammation.

How does NFKBIB (Thr19) phosphorylation status correlate with disease progression in inflammatory and cancer models?

The correlation between NFKBIB (Thr19) phosphorylation status and disease progression represents a significant area for translational research. In inflammatory disease models, increased phosphorylation of NFKBIB at Thr19 often correlates with disease severity due to enhanced NF-κB activation. Research approaches should include tissue microarray analysis of patient samples using phospho-specific antibodies to quantify phosphorylation levels across disease stages . Correlation analysis can then link phosphorylation status with clinical parameters, inflammatory markers, and disease outcomes.

In cancer models, the dysregulation of NF-κB signaling, including altered NFKBIB phosphorylation, is a hallmark of disease progression . Researchers should examine phospho-NFKBIB (Thr19) levels in paired normal and tumor tissues to establish baseline differences. Studies can further stratify samples by cancer stage, grade, and treatment response to identify potential prognostic value. Functional studies using cancer cell lines with modified NFKBIB phosphorylation status (through mutant expression or kinase inhibition) can reveal the impact on proliferation, apoptosis resistance, and metastatic potential. Single-cell analysis techniques provide insights into heterogeneity within tumors, potentially identifying subpopulations with distinct phosphorylation profiles that drive tumor progression. This approach is particularly valuable given the evidence that deregulation of NF-κB and IκB phosphorylations is associated with chronic inflammatory diseases and cancer, making these pathways promising therapeutic targets .

What are the common sources of false positives or negatives when working with phospho-NFKBIB (Thr19) antibodies, and how can they be addressed?

Several technical factors can lead to false results when working with phospho-NFKBIB (Thr19) antibodies. False positives may arise from cross-reactivity with other phosphorylated epitopes, particularly those containing similar phospho-threonine motifs. To address this, researchers should verify antibody specificity using phospho-peptide competition assays, as demonstrated in validation images where signal is blocked by pre-absorption with the immunogen peptide . Antibody validation images confirm that the phospho-specific antibody signal is eliminated when blocked with the phospho-peptide, verifying specificity .

False negatives commonly result from dephosphorylation during sample preparation. This can be prevented by rigorously maintaining cold conditions and including multiple phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers. Inadequate antigen retrieval in immunohistochemistry applications can also lead to false negatives. High-pressure and high-temperature antigen retrieval using Tris-EDTA buffer (pH 8.0) has been validated for effective epitope exposure . Incorrect blocking agents can interfere with antibody binding; using 5% BSA instead of milk (which contains phosphatases) is recommended for phospho-specific antibody applications. For western blot applications, ensure transfer efficiency by using stain-free gel technology or reversible total protein stains before immunoblotting. Including positive controls (TNF-α stimulated cells) and negative controls (phosphatase-treated lysates) in each experiment provides critical reference points for result interpretation.

How can researchers validate the specificity of phospho-NFKBIB (Thr19) antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable results when working with phospho-NFKBIB (Thr19) antibodies. A multi-faceted validation approach should begin with phospho-peptide competition assays, where pre-incubation of the antibody with the phosphorylated immunogen peptide should abolish specific signals in Western blot or immunohistochemistry applications. This approach has been demonstrated in validation studies shown in the search results .

Researchers should also perform comparative analyses using multiple antibodies targeting the same phospho-epitope from different suppliers or clones. Signal concordance across different antibodies strengthens confidence in specificity. Creating phospho-null mutants (T19A) expressed in cells provides an excellent negative control system. Lysates from these cells should show no reactivity with phospho-specific antibodies despite expressing the target protein. Treatment with lambda phosphatase to dephosphorylate samples serves as another negative control approach. For phosphorylation induction validation, stimulate cells with TNF-α (20ng/ml for 30 minutes) to induce phosphorylation, as this has been established as an effective protocol in validation studies .

For mass spectrometry validation, immunoprecipitate NFKBIB using the phospho-specific antibody and confirm the phosphorylation status and site specification using LC-MS/MS analysis. This approach provides direct evidence of antibody specificity for the Thr19 phosphorylation site. Finally, validation across multiple applications (WB, IHC, IF, ELISA) with consistent results significantly strengthens confidence in antibody specificity.

What are the optimal storage and handling conditions to maintain phospho-NFKBIB (Thr19) antibody performance over time?

Proper storage and handling of phospho-NFKBIB (Thr19) antibodies is essential for maintaining antibody performance and experimental reproducibility. Long-term storage should be at -20°C in small aliquots to minimize freeze-thaw cycles, as recommended in product documentation . For antibody formulations containing 50% glycerol (common for many commercial antibodies), avoid freezing at -80°C as this can lead to glycerol stratification and antibody denaturation.

For short-term storage and frequent use within one month, 4°C storage is acceptable according to manufacturer recommendations . When preparing working dilutions, use fresh, high-quality blocking buffer (typically 5% BSA in TBST/PBST) and add sodium azide (0.02%) for preservation if the solution will be stored. Working solutions should generally be used within 1-2 weeks and kept at 4°C. Always centrifuge antibody vials briefly before opening to collect liquid that may have accumulated on the cap or sides.

Quality control measures include periodically testing antibody performance using positive control samples (TNF-α stimulated cells) alongside long-term stored antibody aliquots. Antibody solutions showing signs of precipitation, unusual coloration, or significantly reduced performance should be discarded. When receiving new antibody lots, perform side-by-side comparisons with previous lots to ensure consistent performance. Proper record-keeping of antibody lot numbers, receipt dates, and experimental performance helps track potential degradation over time. Following these storage and handling protocols will help ensure consistent detection of phospho-NFKBIB (Thr19) across experiments and maximize antibody shelf-life.

How does the detection of phospho-NFKBIB (Thr19) compare across different experimental platforms (Western blot, IHC, ELISA)?

Detection of phospho-NFKBIB (Thr19) shows distinct characteristics across different experimental platforms, each with specific advantages and limitations. Western blot analysis provides excellent molecular weight confirmation, with phospho-NFKBIB typically appearing as bands at 48-50 kDa . This technique allows for clear distinction from other potential cross-reactive proteins based on size and is particularly valuable for verifying antibody specificity. Western blotting sensitivity for phospho-NFKBIB (Thr19) is typically sufficient for detecting endogenous levels in stimulated samples, with recommended antibody dilutions ranging from 1:500-1:2000 .

Immunohistochemistry (IHC) offers the advantage of spatial resolution, allowing researchers to visualize phospho-NFKBIB localization within tissue contexts and specific cell types. IHC typically requires higher antibody concentrations (1:100-1:300) compared to Western blotting . The technique is particularly valuable for analyzing phosphorylation status in pathological samples, though it requires careful optimization of antigen retrieval methods. High-pressure and high-temperature Tris-EDTA (pH 8.0) antigen retrieval has been validated for human brain tissue samples .

ELISA provides the highest quantitative precision and throughput capability, making it ideal for processing multiple samples in clinical studies. ELISA typically uses more dilute antibody concentrations (1:5000) and offers better linear range for quantification compared to Western blotting . Cell-based ELISA formats are particularly valuable for screening applications or inhibitor studies. Each platform has complementary strengths, and researchers often benefit from employing multiple techniques in parallel for comprehensive phosphorylation analysis.

What complementary techniques can be combined with phospho-NFKBIB (Thr19) detection to provide a more comprehensive understanding of NF-κB pathway regulation?

A comprehensive understanding of NF-κB pathway regulation requires integrating phospho-NFKBIB (Thr19) detection with complementary techniques. Chromatin immunoprecipitation (ChIP) assays measuring NF-κB binding to target gene promoters can directly link NFKBIB phosphorylation status with transcriptional outcomes. This approach helps establish causality between phosphorylation events and gene expression changes. RNA-seq or qPCR analysis of NF-κB target genes provides functional readouts of pathway activation downstream of NFKBIB phosphorylation.

Proximity ligation assays (PLA) can detect interactions between phospho-NFKBIB and other pathway components, revealing how phosphorylation affects protein-protein interaction networks. Phosphoproteomics using mass spectrometry allows for unbiased identification of multiple phosphorylation sites on NFKBIB and can reveal novel regulatory mechanisms beyond the established Thr19/Ser23 sites. This approach is particularly valuable for understanding the complex interplay between different phosphorylation events.

Live-cell imaging using fluorescent reporters can track NF-κB nuclear translocation in real-time following stimuli that induce NFKBIB phosphorylation. CRISPR-Cas9 gene editing to create phospho-mimetic (T19D) or phospho-null (T19A) mutations provides powerful tools for functional validation. Mathematical modeling incorporating data from these multiple approaches can predict the systems-level consequences of altered NFKBIB phosphorylation under various conditions. This integrative approach overcomes the limitations of any single technique and provides a more holistic understanding of how NFKBIB phosphorylation regulates NF-κB signaling dynamics.

How can phospho-NFKBIB (Thr19) analysis be integrated into multi-parameter flow cytometry assays for single-cell signaling studies?

Integrating phospho-NFKBIB (Thr19) analysis into multi-parameter flow cytometry creates powerful opportunities for single-cell signaling studies. The protocol development begins with fixation and permeabilization optimization, typically using formaldehyde fixation (2-4%) followed by methanol or specialized permeabilization buffers designed for intracellular phospho-epitopes. Researchers should validate antibody performance in flow cytometry using positive controls (TNF-α stimulated cells) and negative controls (phosphatase-treated or T19A mutant-expressing cells).

For panel design, phospho-NFKBIB (Thr19) antibodies should be conjugated to fluorophores with minimal spectral overlap with other key markers. Conjugation can be performed using commercial antibody labeling kits or by purchasing pre-conjugated antibodies if available. The panel should include markers for cell identification (lineage markers), activation status (CD69, CD25), and other relevant phospho-proteins in the NF-κB pathway (phospho-p65, phospho-IKKα/β) to provide pathway context.

Compensation and titration are critical steps, as phospho-specific signals typically have lower intensity ranges than surface markers. Time-course experiments capturing phosphorylation dynamics can reveal heterogeneity in cellular responses within populations. For analysis, traditional gating approaches can be supplemented with dimensionality reduction techniques (tSNE, UMAP) and clustering algorithms to identify cell subpopulations with distinct phosphorylation profiles. This approach is particularly valuable for heterogeneous samples like primary immune cells or tumor specimens where NF-κB signaling may vary dramatically between cell subtypes. Correlation analysis between phospho-NFKBIB (Thr19) and other signaling nodes can reveal network relationships that might not be apparent in population-level studies.

How can researchers effectively use phospho-NFKBIB (Thr19) antibodies to study dysregulated NF-κB signaling in inflammatory disorders?

Studying dysregulated NF-κB signaling in inflammatory disorders requires strategic application of phospho-NFKBIB (Thr19) antibodies across multiple experimental systems. Researchers should begin with comparative analysis of phospho-NFKBIB levels in patient samples versus healthy controls using immunohistochemistry or Western blotting . This baseline difference establishes the relevance of this phosphorylation event in the specific inflammatory condition. For tissue microarray studies, phospho-NFKBIB (Thr19) antibodies can be used at dilutions of 1:100-1:300 with appropriate antigen retrieval methods .

Ex vivo studies with primary cells from patients can reveal altered phosphorylation kinetics in response to inflammatory stimuli. Time-course experiments following TNF-α stimulation (20ng/ml) should be performed to capture both rapid responses (5-30 minutes) and sustained signaling changes (hours to days) . Inhibitor studies targeting specific kinases can help identify dysregulated upstream pathways in patient samples. For animal models of inflammatory diseases, phospho-NFKBIB (Thr19) antibodies can track the efficacy of therapeutic interventions in normalizing NF-κB signaling.

Single-cell approaches combining phospho-flow cytometry with cell type-specific markers can identify which cellular populations exhibit abnormal phospho-NFKBIB regulation within heterogeneous samples. This approach is particularly valuable given that deregulation of NF-κB and IκB phosphorylations is a hallmark of chronic inflammatory diseases, making these constitutively activated signaling pathways promising therapeutic targets . Longitudinal studies correlating phospho-NFKBIB levels with clinical parameters and treatment responses can further establish the biomarker potential of this phosphorylation event in inflammatory disorders.

What experimental designs are most effective for using phospho-NFKBIB (Thr19) detection to evaluate potential inhibitors of the NF-κB pathway?

Designing robust experiments to evaluate NF-κB pathway inhibitors using phospho-NFKBIB (Thr19) detection requires careful consideration of multiple factors. A comprehensive inhibitor screening platform should include both cell-based and biochemical assays. Cell-based screening can utilize cell-based ELISA formats detecting phospho-NFKBIB (Thr19), which allow for higher throughput compared to Western blotting . For these assays, cells should be pre-treated with test compounds at multiple concentrations before stimulation with TNF-α (20ng/ml for 30 minutes) to induce NFKBIB phosphorylation .

Dose-response and time-course experiments are essential to characterize inhibitor potency (IC50 values) and duration of effect. Western blot validation with phospho-NFKBIB (Thr19) antibodies (1:500-1:2000 dilution) provides confirmation of hits identified in screening assays . Including phospho-specific antibodies against multiple NF-κB pathway components (IKKα/β, p65) helps position the inhibitor's mechanism of action within the signaling cascade.

Functional readouts including NF-κB-dependent gene expression (qPCR), reporter assays (luciferase), and phenotypic endpoints (cell survival, cytokine production) should complement the phosphorylation analysis. For in vivo validation, animal models of inflammation or cancer can be treated with lead compounds, and tissues analyzed for phospho-NFKBIB (Thr19) status using immunohistochemistry (1:100-1:300 dilution) . Target engagement can be further confirmed using cellular thermal shift assays (CETSA) or drug affinity responsive target stability (DARTS) approaches. This multi-layered experimental design provides comprehensive evidence for inhibitor efficacy and specificity in modulating the NF-κB pathway through effects on NFKBIB phosphorylation.

How can phospho-NFKBIB (Thr19) analysis contribute to understanding the mechanism of action of anti-inflammatory or anti-cancer therapeutics?

Phospho-NFKBIB (Thr19) analysis provides valuable mechanistic insights into how anti-inflammatory or anti-cancer therapeutics modulate the NF-κB pathway. For mechanism of action studies, researchers should first establish baseline phosphorylation patterns in relevant disease models using Western blotting with phospho-NFKBIB (Thr19) antibodies at validated dilutions (1:500-1:2000) . Time-course experiments tracking phosphorylation changes following drug administration reveal whether a compound acts rapidly (direct kinase inhibition) or requires longer exposure (transcriptional regulation of pathway components).

Comparing phosphorylation patterns across multiple NF-κB pathway nodes helps position the drug's primary site of action. If a compound primarily affects NFKBIB phosphorylation without altering upstream IKK activation, it likely targets the IKK-NFKBIB interaction specifically. For established therapeutics with unknown mechanisms, phospho-NFKBIB analysis can reveal previously unrecognized effects on NF-κB signaling. This approach has identified NF-κB modulation as a secondary mechanism for drugs initially developed to target other pathways.