NFKB2 Antibody

What are the major forms of NFKB2 and their functional significance?

NFKB2 exists in two principal forms: the precursor p100 (97-120 kDa) and the processed active form p52 (52 kDa). The p100 form contains an inhibitory C-terminal domain with ankyrin repeats that must be processed for activation. This processing is a key regulatory step in the non-canonical NF-κB pathway, which controls various immune functions including B cell maturation, antibody production, and T cell function through germinal center regulation .

The p100 form predominantly resides in the cytoplasm, while the processed p52 form translocates to the nucleus where it regulates gene expression. The conversion from p100 to p52 requires phosphorylation, ubiquitination, and partial proteasomal degradation of the C-terminal portion, a process tightly controlled by upstream kinases such as NIK (NF-κB-inducing kinase) .

Mutations in NFKB2 that affect processing can lead to immunodeficiency disorders, highlighting the protein's critical role in immune function .

What applications are suitable for NFKB2 antibodies in research?

NFKB2 antibodies can be utilized across multiple experimental platforms:

These applications allow comprehensive investigation of NFKB2's expression, processing, localization, and function in various experimental contexts .

How do different NFKB2 antibodies compare in terms of epitope recognition and functional applications?

NFKB2 antibodies target different regions of the protein, affecting their utility in specific applications:

When selecting an antibody, researchers should consider the specific research question. For monitoring pathway activation, antibodies detecting both forms are preferable. For mechanistic studies of processing, combining N-terminal and C-terminal antibodies provides more complete information .

What are optimal protocols for detecting both p100 and p52 forms by Western blot?

Successful detection of both NFKB2 forms requires careful optimization:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors when studying phosphorylated forms

  • Prepare both cytoplasmic (20 μg) and nuclear (10 μg) fractions for comprehensive analysis

• Gel selection and electrophoresis:

  • Use gradient gels (4-12%) to resolve both high (p100) and low (p52) molecular weight forms

  • Run at lower voltage (80-100V) for better resolution of both forms

• Transfer conditions:

  • Use wet transfer for efficient transfer of both proteins

  • Consider semi-dry transfer with graduated buffer systems for the large p100 protein

  • Transfer at 30V overnight at 4°C for optimal results with both forms

• Membrane blocking:

  • Block with 5% BSA in TBST (preferred over milk for phosphorylated proteins)

  • Incubate 1 hour at room temperature or overnight at 4°C

• Antibody incubation:

  • Use antibodies that recognize epitopes present in both forms

  • Typical dilutions range from 1:1000-1:2000 in 5% BSA

  • Incubate primary antibody overnight at 4°C

• Detection strategy:

  • Consider different exposure times to visualize both bands optimally

  • Use enhanced chemiluminescence or fluorescent secondary antibodies for sensitive detection

This optimized protocol allows reliable detection of both p100 and p52 forms, enabling accurate assessment of processing activity .

How should NFKB2 antibodies be validated for experimental use?

Thorough validation is essential for obtaining reliable results with NFKB2 antibodies:

• Positive controls:

  • Cell lines with known NFKB2 expression:

    • Daudi and Raji (human Burkitt's lymphoma)

    • Jurkat (human T cell leukemia)

    • HeLa, K-562, NIH/3T3, RAW 264.7, U2OS

  • Stimulated samples showing increased p52 generation:

    • CD40 ligand treatment (100 ng/mL for 4 hours)

    • PMA (50 ng/mL) with calcium ionomycin (200 ng/mL)

• Negative controls:

  • NFKB2 knockout or knockdown samples

  • Isotype control antibodies for immunostaining

  • Preimmune serum for polyclonal antibodies

• Specificity assays:

  • Western blot showing bands at expected molecular weights (52 kDa and 100-120 kDa)

  • Peptide competition assays with immunizing peptide

  • Multiple antibody comparison using antibodies targeting different epitopes

  • Stimulation experiments showing dynamic changes in p100/p52 ratio

• Cross-reactivity assessment:

  • Test across species if cross-reactivity is claimed

  • Verify minimal cross-reactivity with related proteins (NFKB1/p50)

  • Note: Some antibodies work with human and mouse but not all work with rat samples

• Application-specific validation:

  • For IHC: Test multiple antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • For ChIP: Verify enrichment at known NF-κB target genes

  • For IP: Confirm pull-down of interaction partners

Proper validation ensures experimental results accurately reflect biological processes rather than technical artifacts .

What stimulation protocols effectively induce NFKB2 processing for experimental studies?

The non-canonical NF-κB pathway can be activated through several stimulation protocols:

• CD40 ligand treatment:

  • Concentration: 100 ng/mL recombinant human CD40L/TNFSF5

  • Duration: 4 hours (optimal for initial processing studies)

  • Suitable for B cell lines (e.g., Daudi, Raji)

  • Results in increased p52 levels with corresponding decrease in p100

• PMA with calcium ionomycin:

  • Concentrations: 50 ng/mL PMA + 200 ng/mL calcium ionomycin

  • Duration: Overnight incubation (12-16 hours)

  • Effective for T cell lines (e.g., Jurkat)

  • Induces strong activation of NF-κB pathways

• Anti-CD3 stimulation:

  • Method: Plate-bound or soluble anti-CD3 antibody (clone OKT3)

  • Duration: 2 days for optimal processing

  • Appropriate for primary T cells and some T cell lines

  • Enables assessment of TCR-mediated NF-κB activation

• BAFF (B cell activating factor) treatment:

  • Concentration: 50-100 ng/mL

  • Duration: 6-24 hours (kinetics may vary by cell type)

  • Particularly effective for B cells

  • Specifically activates the non-canonical pathway

• Lymphotoxin-β receptor agonists:

  • Concentration: 1-2 μg/mL anti-LTβR antibody

  • Duration: 12-24 hours

  • Effective in many cell types including fibroblasts and epithelial cells

When analyzing pathway activation, a time course experiment is recommended, as processing kinetics can vary by cell type and stimulus. Nuclear and cytoplasmic fractionation provides additional information about p52 translocation following processing .

How can NFKB2 antibodies be employed in chromatin immunoprecipitation studies?

Chromatin immunoprecipitation (ChIP) with NFKB2 antibodies allows identification of genomic binding sites, providing insights into gene regulation. A detailed protocol based on search results includes:

• Cell preparation:

  • Culture cells of interest (e.g., Jurkat cells)

  • Stimulate with appropriate activators of the non-canonical pathway:

    • PMA (50 ng/mL) and calcium ionomycin (200 ng/mL) overnight

    • CD40 ligand for B cells (100 ng/mL)

• Crosslinking and chromatin preparation:

  • Fix cells with 1% formaldehyde (10 minutes at room temperature)

  • Quench with 125 mM glycine

  • Lyse cells and sonicate to generate DNA fragments (200-500 bp)

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with NFKB2 antibody (5 μg per IP reaction)

  • Include appropriate controls (IgG isotype control)

  • For enhanced sensitivity, consider the ultrasonic bath technique mentioned in search result

• Capture and elution:

  • Add biotinylated secondary antibody

  • Capture complexes using streptavidin ferrofluid (50 μL)

  • Wash extensively to remove non-specific binding

  • Elute protein-DNA complexes

• Reverse crosslinking and DNA purification:

  • Incubate at 65°C overnight to reverse formaldehyde crosslinks

  • Treat with proteinase K

  • Purify DNA with chelating resin solution or commercial kits

• Analysis options:

  • qPCR: For known target genes (e.g., NFKB2-regulated genes)

  • ChIP-seq: For genome-wide binding profile analysis

  • CUT&RUN or CUT&Tag: For higher sensitivity and lower background

This approach has revealed NFKB2 binding to various target genes involved in immune regulation, inflammation, and cell survival .

What are the methodological approaches for studying NFKB2-related immunodeficiency disorders?

Investigating NFKB2-related immunodeficiency disorders requires multiple complementary approaches:

• Protein processing analysis:

  • Western blotting to assess p100 processing to p52

  • Compare patient samples with healthy controls following stimulation

  • Analyze both unstimulated and stimulated conditions (e.g., anti-CD3 for 2 days)

  • Quantify p100/p52 ratio as indicator of processing defects

  • Example finding: "Patient's PBMCs exhibited increased p100 levels and reduced p52 levels, indicative of impaired cleavage of p100 into its active form"

• Genetic-functional correlation:

  • Whole exome sequencing to identify NFKB2 variants

  • Functional classification of variants:

    • p52 LOF/IκBδ GOF: Processing defects (most common)

    • p52 LOF/IκBδ LOF: Haploinsufficiency

    • p52 GOF/IκBδ LOF: Gain-of-function

  • Structural modeling to predict variant impact

  • Example: "Structural modeling indicates that the Thr684Pro variant causes steric clash"

• Cellular immunophenotyping:

  • Flow cytometry to assess B and T cell subsets

  • Evaluate memory B cell populations (often reduced)

  • Monitor CD19+ B cell counts (frequently low)

  • Assess T helper and regulatory T cell function

• Immunological parameter assessment:

  • Measure serum immunoglobulin levels

  • Evaluate antibody responses to vaccination

  • Test for presence of autoantibodies (including anti-interferon autoantibodies)

  • Example finding: "Whole-exome sequencing identified a missense variant in the ankyrin domain of NFKB2... The patient's pan-hypogammaglobulinemia, anti-IFN-α2 autoantibodies, and susceptibility to severe viral infections indicate the deleterious effect"

• Functional genomics:

  • RNA sequencing to identify dysregulated genes

  • Compare gene expression profiles between patients and controls

  • Pathway analysis to identify affected biological processes

These methodological approaches have revealed that NFKB2 mutations can lead to diverse clinical phenotypes, including CVID, combined immunodeficiency, autoimmunity, endocrinopathy, and increased susceptibility to viral infections .

How can NFKB2 antibodies be utilized to differentiate between canonical and non-canonical NF-κB pathway activation?

Differentiating between canonical and non-canonical NF-κB pathway activation is critical for understanding specific signaling mechanisms. NFKB2 antibodies play a key role in this differentiation:

• Protein marker distinction:

  • Canonical pathway: Monitor p105/p50 (NFKB1), IκBα, and RelA (p65)

  • Non-canonical pathway: Track p100/p52 (NFKB2) and RelB

  • Use specific antibodies for each protein component

  • Create a panel approach integrating multiple markers

• Kinetic analysis:

  • Canonical pathway: Rapid activation (minutes to hours)

  • Non-canonical pathway: Delayed activation (hours to days)

  • Design time-course experiments (0, 15, 30 min, 1, 2, 4, 8, 24 hours)

  • Use NFKB2 antibodies to track p100 processing to p52 over time

• Subcellular fractionation:

  • Prepare nuclear and cytoplasmic fractions separately

  • As described: "Gels were loaded with 20 μg of cytoplasmic (Cyto) and 10 μg of nuclear extracts (Nuc)"

  • Nuclear accumulation of p52 indicates non-canonical activation

  • Cytoplasmic reduction of p100 confirms processing

• Stimulus-specific experimental design:

  • Canonical pathway activators: TNF-α, IL-1, LPS

  • Non-canonical pathway activators: CD40L, BAFF, lymphotoxin-β

  • Example: "Daudi human Burkitt's lymphoma cell line untreated (-) or treated (+) with 100 ng/mL Recombinant Human CD40 Ligand/TNFSF5"

  • Parallel stimulation with pathway-specific activators provides comparative data

• Combined detection methodologies:

  • Western blotting: For processing and expression level changes

  • ChIP-seq: For binding site specificity differences

  • Immunofluorescence: For visualization of nuclear translocation

  • Flow cytometry: For single-cell analysis of pathway activation

• Genetic approach:

  • Use cells with specific knockdown of pathway components:

    • siRNA against NFKB2 to impair non-canonical signaling

    • siRNA against NFKB1 to disrupt canonical signaling

    • Compare effects on downstream targets

This multi-faceted approach allows clear delineation between these interconnected but distinct signaling pathways, facilitating more precise mechanistic studies .

What factors affect the p100/p52 ratio in experimental samples and how should they be interpreted?

The p100/p52 ratio serves as a critical readout of non-canonical NF-κB pathway activity. Multiple factors influence this ratio, requiring careful interpretation:

• Biological factors influencing the ratio:

  • Basal activation state of cells (resting vs. activated)

  • Cell type-specific processing rates

  • Tissue microenvironment and external stimuli

  • Genetic variations affecting processing machinery

• Interpretation of p100/p52 ratio patterns:

• Technical considerations affecting ratio measurement:

  • Antibody selection (epitope location affects detection)

  • Sample preparation (nuclear vs. cytoplasmic fractionation)

  • Time point selection (processing is dynamic)

  • Protein extraction efficiency

• Experimental controls for accurate interpretation:

  • Include positive controls (stimulated samples)

  • Run time-course experiments to capture processing dynamics

  • Compare with known NFKB2 mutation samples when available

  • Use appropriate loading controls (β-actin for cytoplasmic, lamin for nuclear)

Understanding these patterns is particularly relevant for diagnosing NFKB2-related immunodeficiencies, where processing defects lead to characteristic alterations in the p100/p52 ratio .

What are the most common technical challenges in NFKB2 antibody applications and their solutions?

Researchers working with NFKB2 antibodies encounter several technical challenges that require specific solutions:

• Western blotting challenges:

• Immunohistochemistry/Immunofluorescence challenges:

• ChIP and related techniques:

• Flow cytometry challenges:

These solutions derive from experimental approaches described in the search results and represent best practices for obtaining reliable data with NFKB2 antibodies .

How can researchers address discrepancies between NFKB2 antibody results and functional data?

When NFKB2 antibody results conflict with functional data, a systematic troubleshooting approach is essential:

• Antibody validation reassessment:

  • Confirm antibody specificity with additional controls

  • Test multiple antibodies targeting different epitopes

  • Verify recognition of both human and mouse proteins if working across species (note the 96% amino acid sequence identity mentioned in )

  • Include knockout/knockdown validation if available

• Technical vs. biological variability analysis:

  • Replicate experiments to distinguish random variation from true discrepancies

  • Standardize protocols across experiments (stimulation times, antibody concentrations)

  • Document lot-to-lot antibody variation

  • Consider genetic differences between cell lines or primary samples

• Post-translational modification considerations:

  • Phosphorylation status affects processing and detection

  • Some antibodies may be sensitive to specific modifications

  • Test phospho-specific antibodies (e.g., pSer866, pSer870 mentioned in )

  • Compare results under different stimulation conditions

• Integrative data analysis approach:

  • Combine protein (Western blot) with mRNA (qPCR, RNA-seq) analysis

  • Correlate with downstream functional readouts (target gene expression)

  • Use multiple technical approaches (Western blot, IHC, flow cytometry)

  • Example integration: "Whole-exome sequencing identified a missense variant in the ankyrin domain of NFKB2... PBMCs exhibited impaired cleavage of p100"

• Mechanistic resolution strategies:

  • For processing discrepancies: Examine NIK and IKKα activity

  • For nuclear translocation issues: Verify nuclear import machinery

  • For target gene differences: Assess cofactor availability

  • For patient samples: Consider mutational effects on epitope recognition

• Experimental design refinement:

  • Include appropriate time points (non-canonical pathway has delayed kinetics)

  • Test multiple stimulation conditions

  • Use fractionation to distinguish subcellular pools

  • When working with genetic variants, express the mutant protein in control cells

• Consideration of alternative explanations:

  • Compensatory mechanisms in genetic models

  • Non-transcriptional functions of NFKB2

  • Alternative processing pathways

  • Cell type-specific regulatory mechanisms

This systematic approach has successfully resolved discrepancies in NFKB2 studies, particularly in cases of immunodeficiency where protein detection data initially appeared contradictory to clinical phenotypes .

How do recent findings on NFKB2 mutations inform antibody selection and experimental design?

Recent research has identified distinct categories of NFKB2 mutations with specific functional consequences, significantly impacting antibody selection and experimental approaches:

• Mutation classification and corresponding antibody strategy:

• Domain-specific mutation analysis necessitates targeted detection:

  • Ankyrin domain mutations: "Thr684Pro variant causes steric clash"

  • C-terminal degron mutations: Affect phosphorylation sites required for processing

  • Truncating mutations: May eliminate epitopes in C-terminal region

• Experimental design considerations based on mutation location:

  • For C-terminal mutations: Include phosphorylation analysis

  • For ankyrin domain mutations: Assess protein-protein interactions

  • For N-terminal mutations: Evaluate DNA binding capacity

• Stimulation protocol refinement:

  • Processing-defective mutations: Extend stimulation time to detect any residual processing

  • Haploinsufficiency: Quantitative comparison with heterozygous controls

  • Gain-of-function: Include repression/negative feedback assessment

• Clinical-research correlation approaches:

  • Autoantibody studies: "Patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies"

  • Immune cell abnormalities: "low memory B cells were more common in p52 LOF/IκBδ GOF patients"

  • Viral susceptibility: "Patients with NFKB2 mutations, particularly p52 LOF/IκBδ GOF, are at higher risk of viral infections"

• Emerging genotype-phenotype correlations:

  • p52 LOF/IκBδ GOF: Associated with autoantibodies against type I IFNs

  • Haploinsufficiency: Variable penetrance, milder phenotype

  • Different mutation types correlate with specific clinical features (endocrinopathy, ectodermal dysplasia)

These findings emphasize the importance of comprehensive mutation characterization when studying NFKB2-related disorders and highlight the need for multiple detection approaches to fully assess functional impacts .

What novel methodologies are emerging for studying NFKB2 dynamics and interactions?

Recent technological advances have expanded the toolkit for studying NFKB2 dynamics and interactions:

• Advanced chromatin profiling techniques:

  • CUT&RUN: Enhanced sensitivity over traditional ChIP

    • Recommended NFKB2 antibody dilution: 1:50

    • Enables genome-wide binding profiles with lower background

    • Requires fewer cells than conventional ChIP

  • CUT&Tag: In situ chromatin profiling

    • Also uses 1:50 antibody dilution

    • Allows single-cell resolution of chromatin binding

    • Preserves nuclear architecture during analysis

• Proximity-based protein interaction detection:

  • BioID/TurboID: Proximity labeling for identifying interaction partners

    • Fusion of biotin ligase to NFKB2 identifies neighbors in living cells

    • Can distinguish p100 from p52 interactomes

  • APEX2: Spatial proteomics for subcellular interaction mapping

    • Higher temporal resolution than BioID

    • Useful for capturing dynamic changes during processing

• Live-cell imaging approaches:

  • CRISPR-mediated endogenous tagging of NFKB2

    • Maintains physiological expression levels

    • Allows real-time monitoring of processing and localization

  • Split fluorescent protein complementation

    • Visualizes specific NFKB2 interactions in living cells

    • Can distinguish between p100 and p52 interactions

• Single-cell technologies:

  • Single-cell RNA-seq combined with protein detection

    • Correlates NFKB2 protein levels with transcriptional output

    • Reveals cell-to-cell variability in pathway activation

  • Mass cytometry (CyTOF)

    • Simultaneously measures multiple NF-κB pathway components

    • Allows detailed phenotyping of immune cell subsets in patient samples

• Structural biology approaches:

  • Cryo-EM of NFKB2 complexes

    • Reveals conformational changes during processing

    • Helps interpret effects of disease-associated mutations

  • Hydrogen-deuterium exchange mass spectrometry

    • Maps dynamic protein regions during activation

    • Identifies surfaces involved in protein-protein interactions

• Functional genomics techniques:

  • CRISPR screening for NFKB2 pathway components

    • Identifies novel regulators of processing

    • Screens for synthetic interactions with NFKB2 mutations

  • RNA-seq analysis in patient samples

    • As employed in search result for patient characterization

    • Reveals dysregulated gene networks downstream of NFKB2

These emerging methodologies provide unprecedented insights into NFKB2 biology and are particularly valuable for understanding disease mechanisms in immunodeficiency disorders .

What are current best practices for NFKB2 antibody selection and experimental design?

Based on the comprehensive review of available research, the following best practices emerge for NFKB2 antibody applications:

• Strategic antibody selection:

  • For total NFKB2 detection: Choose antibodies recognizing epitopes present in both p100 and p52 forms

  • For specific processing analysis: Combine N-terminal and C-terminal targeting antibodies

  • For activation studies: Include phospho-specific antibodies

  • Validate across multiple applications if using for different techniques

  • Consider species specificity needs (human, mouse, rat compatibility)

• Experimental design principles:

  • Include positive controls (stimulated samples showing processing)

  • Implement proper negative controls (isotype antibodies, blocking peptides)

  • Design time-course experiments to capture processing kinetics

  • Prepare both nuclear and cytoplasmic fractions for comprehensive analysis

  • Use pathway-appropriate stimuli (CD40L, BAFF, anti-CD3)

• Validation and quality control:

  • Verify detection of bands at correct molecular weights (52 kDa and 100-120 kDa)

  • Test multiple antibody dilutions to optimize signal-to-noise ratio

  • Confirm specificity with knockout or knockdown samples when available

  • Use multiple antibodies targeting different epitopes for critical findings

  • Document antibody lot information for reproducibility

• Application-specific considerations:

  • Western blot: Use gradient gels, optimize transfer for high molecular weight p100

  • IHC: Compare TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

  • ChIP: Use 5 μg antibody per IP, stimulate cells appropriately

  • Flow cytometry: Optimize fixation and permeabilization protocols

• Data interpretation guidelines:

  • Analyze p100/p52 ratio changes rather than absolute levels

  • Consider cell type-specific baseline processing rates

  • Interpret results in context of pathway activation status

  • Account for mutation effects when analyzing patient samples

  • Correlate protein data with functional readouts

By adhering to these evidence-based best practices, researchers can generate reliable, reproducible data that advances understanding of NFKB2 biology and related disorders .

How can researchers ensure reproducibility and reliability in NFKB2 antibody-based experiments?

Ensuring reproducibility and reliability in NFKB2 antibody-based experiments requires systematic attention to multiple factors:

• Comprehensive antibody documentation:

  • Record complete antibody information:

    • Catalog number and manufacturer (e.g., "Mouse Anti-Human NF kappa B2 Monoclonal Antibody (Catalog # MAB28881)" )

    • Clone number for monoclonals (e.g., "Clone # 291319" )

    • Lot number to track lot-to-lot variation

    • Epitope information (e.g., "AA 1-340" )

  • Validate reactivity across species used in your research

  • Document storage conditions and handling protocols

• Standardized experimental protocols:

  • Establish detailed SOPs for each application

  • Specify critical parameters:

    • Antibody concentrations (e.g., "0.5 µg/mL" for WB)

    • Incubation times and temperatures

    • Buffer compositions ("Immunoblot Buffer Group 1" )

    • Antigen retrieval methods ("TE buffer pH 9.0" )

  • Include positive and negative controls in every experiment

• Cell and tissue preparation consistency:

  • Standardize cell culture conditions

  • Document passage number and confluence

  • Use consistent stimulation protocols

    • "100 ng/mL Recombinant Human CD40 Ligand/TNFSF5 for 4 hours"

    • "PMA (50 ng/mL) and calcium ionomycin (200 ng/mL) overnight"

  • Prepare nuclear and cytoplasmic fractions using consistent methods

• Quantification and data analysis practices:

  • Use digital image capture with linear dynamic range

  • Implement standardized quantification methods

    • "Quantified by ImageJ2 software"

  • Normalize to appropriate loading controls

  • Apply consistent statistical approaches

• Validation across multiple approaches:

  • Verify key findings with orthogonal methods

  • Combine protein and mRNA analysis

  • Support antibody-based findings with functional assays

  • For patient studies, integrate with genetic and clinical data

• Reporting standards:

  • Adhere to complete reporting guidelines

  • Include detailed methods sections with all parameters

  • Publish raw data when possible

  • Document limitations and potential sources of variability

• Quality control measures:

  • Regular antibody validation

  • Testing of new antibody lots

  • Inclusion of inter-assay controls

  • Participation in standardization initiatives

Following these guidelines significantly improves reliability and reproducibility, particularly in challenging contexts such as patient sample analysis where limited material demands optimal experimental design .