NFKBIA Antibody

What is NFKBIA and why is it important in cellular research?

NFKBIA (Nuclear Factor of kappa Light Polypeptide Gene Enhancer in B-Cells Inhibitor, alpha) is a critical regulator of the NF-κB pathway, one of the most important signaling cascades in inflammation and immune responses. It functions by inhibiting the activity of dimeric NF-kappa-B/REL complexes by trapping REL (RELA/p65 and NFKB1/p50) dimers in the cytoplasm through masking their nuclear localization signals . Upon cellular stimulation by immune and pro-inflammatory responses, NFKBIA becomes phosphorylated (primarily at serine residues 32 and 36), which promotes its ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription .

Research on NFKBIA is particularly relevant because mutations in this gene have been associated with ectodermal dysplasia anhidrotic with T-cell immunodeficiency autosomal dominant disease . Additionally, haploinsufficient deletions of NFKBIA have been identified as significant prognostic markers in gliomas, correlating with poor patient outcomes .

How should I select the appropriate NFKBIA antibody for my experiment?

Selection of the appropriate NFKBIA antibody depends on several experimental considerations:

• Target region specificity: Determine whether you need antibodies targeting specific domains (N-terminal, C-terminal) or particular amino acid sequences. For example, some antibodies target AA 1-317 , N-Term , AA 12-41 , or specific phosphorylation sites (pSer32/pSer36) .

• Species reactivity: Confirm the antibody reacts with your species of interest. Most NFKBIA antibodies are reactive against human samples, but many also cross-react with mouse and rat samples .

• Application compatibility: Verify the antibody is validated for your specific application:

  • Western Blotting (WB)

  • Immunohistochemistry (IHC)

  • Immunofluorescence (IF)

  • ELISA

  • Flow Cytometry (FACS)

  • Immunocytochemistry (ICC)

• Validation evidence: Look for antibodies with published validation data. For example, some NFKBIA antibodies have been cited in multiple research publications, indicating reliability .

Table 1: Common recommended dilutions for NFKBIA antibodies in different applications

What controls should I include when using NFKBIA antibodies in Western blotting?

When performing Western blotting with NFKBIA antibodies, these controls are critical for valid interpretation:

• Positive control: Include cell lines known to express NFKBIA, such as Raji human Burkitt's lymphoma cell line .

• Stimulated/unstimulated pairs: For phospho-specific antibodies (e.g., those targeting pSer32/pSer36), include both stimulated samples (e.g., with TNF-α or IL-1β) and unstimulated controls to demonstrate phosphorylation-dependent recognition.

• Blocking peptide control: Include samples where the antibody is pre-incubated with the immunizing peptide to verify specificity.

• Loading control: Use housekeeping proteins (GAPDH, β-actin) to ensure equal loading across lanes.

• Molecular weight markers: NFKBIA typically appears at approximately 37-40 kDa, though this may vary based on post-translational modifications.

• siRNA or CRISPR knockout controls: When available, include samples where NFKBIA has been knocked down or knocked out to confirm antibody specificity.

How can phospho-specific NFKBIA antibodies be used to study NF-κB pathway dynamics?

Phospho-specific antibodies targeting Ser32/Ser36 of NFKBIA are powerful tools for studying the temporal dynamics of NF-κB pathway activation:

• Time-course experiments: After stimulation with NF-κB activators (TNF-α, IL-1β, LPS), samples collected at different time points can be analyzed using phospho-specific antibodies to track NFKBIA phosphorylation, which precedes its degradation .

• Pathway inhibitor studies: The effects of pathway inhibitors (e.g., IKK inhibitors) can be quantitatively assessed by measuring changes in NFKBIA phosphorylation status.

• Single-cell analysis: Phospho-specific antibodies can be used in flow cytometry or immunofluorescence to analyze heterogeneity in pathway activation at the single-cell level.

• Stimulus-specific responses: Different stimuli may lead to distinct phosphorylation kinetics, which can be revealed using phospho-specific antibodies.

• Mathematical modeling: Quantitative data obtained using phospho-specific antibodies can inform mathematical models of NF-κB signaling dynamics.

The key advantage of phospho-specific antibodies is their ability to detect the activation state of the pathway rather than merely the presence of the protein. This is particularly important given that NFKBIA inhibits NF-κB signaling in its unphosphorylated state but promotes pathway activation once phosphorylated .

What methodological considerations are important when studying NFKBIA deletions in cancer samples?

NFKBIA deletions have emerged as important prognostic markers in gliomas, with haploinsufficient deletions associated with poor outcomes . When investigating these deletions:

• Deletion detection methods:

  • Use a combination of techniques (FISH, qPCR, next-generation sequencing) to confirm deletions

  • Account for heterogeneity within tumor samples

  • Distinguish focal deletions (involving only NFKBIA) from larger chromosomal losses

• Integration with other genetic markers:

  • Analyze NFKBIA deletions in the context of other genetic alterations (IDH mutations, 1p19q codeletions, TERT mutations)

  • Consider potential interactions between NFKBIA deletions and other alterations

• Tumor progression analysis:

  • Compare primary and recurrent tumors to assess acquisition of NFKBIA deletions during disease progression

  • Research suggests NFKBIA deletions are more frequent in recurrent tumors (31.0%) than in primary tumors (16.1%)

• Functional validation:

  • Use NFKBIA antibodies to confirm protein loss in deletion-positive samples

  • Perform pathway activation studies to assess the functional consequences of NFKBIA deletions

• Epigenetic correlates:

  • Investigate DNA methylation patterns associated with NFKBIA deletions

  • Research has shown NFKBIA deletions correlate with hypomethylation at specific CpG sites

How should researchers optimize immunohistochemistry protocols for NFKBIA detection in FFPE tissue samples?

Detecting NFKBIA in formalin-fixed paraffin-embedded (FFPE) tissues requires careful optimization:

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Optimize retrieval duration and temperature

  • Consider protein cross-linking effects from formalin fixation

• Antibody selection and validation:

  • Choose antibodies specifically validated for IHC in FFPE tissues

  • Test antibodies at multiple concentrations (typically 1:50-1:200)

  • Validate staining patterns with positive and negative control tissues

• Signal amplification considerations:

  • Evaluate need for signal amplification systems (polymer-based detection, tyramide signal amplification)

  • Balance signal strength with background staining

• Counterstaining and visualization:

  • Optimize nuclear counterstaining to allow clear visualization of NFKBIA's subcellular localization

  • Consider dual immunofluorescence to colocalize NFKBIA with other pathway components

• Quantification approaches:

  • Develop consistent scoring systems (H-score, Allred score)

  • Consider digital pathology methods for more objective quantification

  • Account for both nuclear and cytoplasmic staining in analysis

How can NFKBIA antibodies be used in chromatin immunoprecipitation studies?

NFKBIA has nuclear functions in addition to its cytoplasmic role, including interactions with histones H2A and H4 to regulate polycomb-dependent transcriptional repression . Chromatin immunoprecipitation (ChIP) using NFKBIA antibodies can reveal:

• Protocol optimization:

  • Fix cells using formaldehyde (typically 1% for 10 minutes)

  • Sonicate chromatin to appropriate fragment size (200-500 bp)

  • Use 5-10 μg of NFKBIA antibody per IP reaction

  • Include appropriate controls (IgG control, input DNA)

• Protein-DNA interaction analysis:

  • Use NFKBIA antibodies to pull down chromatin regions where NFKBIA is bound

  • Perform qPCR or sequencing on immunoprecipitated DNA to identify binding sites

  • Example protocol: Jurkat cells treated with PMA and calcium ionomycin, fixed with formaldehyde, and sonicated can be used for NFKBIA ChIP

• Interaction with histone modifications:

  • Perform sequential ChIP (Re-ChIP) to identify regions where NFKBIA co-occurs with specific histone modifications

  • Investigate NFKBIA's role in regulating chromatin structure and gene expression

• Stimulus-dependent chromatin binding:

  • Compare NFKBIA chromatin binding before and after cellular stimulation

  • Identify dynamic changes in NFKBIA's nuclear interactions

What are potential causes of non-specific bands when using NFKBIA antibodies in Western blotting?

Non-specific bands are a common challenge with NFKBIA antibodies. Here are methodological approaches to address this issue:

• Cross-reactivity with NFKBIA family members:

  • NFKBIA has structural similarity with other IκB family proteins (NFKBIB, NFKBIE)

  • Use blocking peptides specific to NFKBIA to confirm band specificity

  • Consider using knockout or knockdown controls

• Post-translational modifications:

  • NFKBIA undergoes phosphorylation, ubiquitination, and other modifications

  • Different bands may represent modified forms of NFKBIA

  • Treatment with phosphatases or deubiquitinating enzymes can confirm modification-dependent banding patterns

• Proteolytic fragments:

  • NFKBIA can be cleaved by proteases, generating fragments

  • Use protease inhibitors during sample preparation

  • Compare patterns between fresh and degraded samples

• Antibody optimization:

  • Test different concentrations (typically 0.5-1 μg/mL for Western blot)

  • Optimize blocking solutions (5% non-fat milk vs. BSA)

  • Adjust washing stringency and duration

• Sample preparation considerations:

  • Ensure complete protein denaturation

  • Use fresh samples and avoid freeze-thaw cycles

  • Consider alternative lysis buffers to improve specificity

How can researchers address discrepancies between NFKBIA protein levels and gene expression data?

Researchers often observe mismatches between NFKBIA protein levels (detected by antibodies) and mRNA expression. To methodologically address these discrepancies:

• Post-transcriptional regulation:

  • Measure NFKBIA mRNA stability (actinomycin D chase experiments)

  • Investigate microRNA-mediated regulation of NFKBIA

  • Examine RNA-binding protein interactions affecting NFKBIA mRNA

• Post-translational regulation:

  • Assess protein stability using cycloheximide chase experiments

  • Measure ubiquitination status using specific ubiquitin antibodies

  • Investigate proteasomal degradation with inhibitors (MG132)

• Temporal considerations:

  • Perform time-course experiments to identify delays between mRNA induction and protein accumulation

  • Account for NFKBIA's rapid turnover in stimulated cells

• Technical validation:

  • Use multiple antibodies targeting different epitopes

  • Confirm antibody specificity with knockdown/knockout controls

  • Validate mRNA measurements with multiple primer sets or techniques

• Single-cell analyses:

  • Consider cell population heterogeneity

  • Use single-cell techniques to correlate mRNA and protein at the individual cell level

How can NFKBIA antibodies be used to study immunodeficiency disorders associated with NFKBIA mutations?

Germline heterozygous gain-of-function mutations in NFKBIA cause an autosomal dominant form of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) . Researching these conditions with NFKBIA antibodies requires:

• Mutation-specific considerations:

  • Design experiments to detect both wild-type and mutant NFKBIA

  • Consider using antibodies targeting regions away from common mutation sites

  • For phospho-specific antibodies, be aware that GOF mutations often prevent phosphorylation on serine 32 or 36

• Functional assays:

  • Measure NF-κB pathway activation in patient cells using phospho-specific antibodies

  • Assess NFKBIA degradation kinetics following cellular stimulation

  • Compare nuclear vs. cytoplasmic localization of NF-κB components

• Therapeutic monitoring:

  • Use NFKBIA antibodies to monitor effects of treatments (e.g., hematopoietic stem cell transplantation)

  • Quantify changes in protein levels and phosphorylation status

• Cell type-specific analyses:

  • Compare NFKBIA regulation across different immune cell populations

  • Correlate NFKBIA dysfunction with cell-specific functional defects

What are best practices for using NFKBIA antibodies to study its role in glioma progression?

NFKBIA deletions reshape the epigenome and are associated with poor prognosis in gliomas . When investigating NFKBIA in glioma:

How might NFKBIA antibodies be incorporated into multiplexed imaging approaches?

As multiplexed tissue imaging technologies advance, NFKBIA antibodies can be integrated into comprehensive analyses:

• Multiplex immunofluorescence:

  • Combine NFKBIA antibodies with antibodies targeting other pathway components

  • Use spectrally distinct fluorophores to visualize multiple targets simultaneously

  • Implement sequential staining protocols for higher-order multiplexing

• Mass cytometry approaches:

  • Conjugate NFKBIA antibodies with metal isotopes for CyTOF or Imaging Mass Cytometry

  • Simultaneously detect dozens of proteins including NFKBIA and related pathway components

  • Perform high-dimensional analysis of spatial relationships

• Digital spatial profiling:

  • Incorporate NFKBIA antibodies into oligonucleotide-tagged antibody panels

  • Perform region-specific quantification in heterogeneous tissues

  • Correlate NFKBIA with spatially restricted gene expression patterns

• Quality control considerations:

  • Validate antibody performance in multiplexed formats

  • Test for spectral overlap or antibody interference

  • Include appropriate single-stain controls