NFKB2 (Ab-870) Antibody

What is NFKB2 (Ab-870) Antibody and what specific epitope does it recognize?

NFKB2 (Ab-870) Antibody is a polyclonal antibody that specifically recognizes the phosphorylated serine residue at position 870 (pSer870) in the human Nuclear Factor NF-kappa-B p100 subunit (NFKB2) . This antibody detects endogenous levels of NFKB2 only when phosphorylated at Serine 870, making it highly specific for the activated form of this protein . The epitope corresponds to a peptide sequence around aa.868~872 (Y-G-S-Q-S) derived from Human NF-kB-p100 . It's important to note that the phosphorylation position varies slightly between species: in humans it's S870, in mice S869, and in rats S868 .

Which experimental applications is the NFKB2 (Ab-870) Antibody validated for?

The NFKB2 (Ab-870) Antibody has been validated for multiple experimental applications:

The antibody is particularly useful for Western blotting applications to detect phosphorylated NFKB2 in cell or tissue lysates . For optimal results in immunohistochemistry applications, researchers should follow the recommended protocol using paraffin-embedded sections (IHC-P) .

What species reactivity has been confirmed for NFKB2 (Ab-870) Antibody?

The NFKB2 (Ab-870) Antibody has been confirmed to react with human, mouse, and rat samples . Additional predicted reactivity (based on sequence homology but not necessarily experimentally confirmed) includes:

• Pig

• Bovine

• Horse

• Sheep

• Rabbit

• Dog

• Chicken

This broad cross-reactivity makes the antibody suitable for comparative studies across multiple model organisms, though validation in your specific experimental system is always recommended.

How should I optimize experimental conditions when using NFKB2 (Ab-870) Antibody?

For optimal results with NFKB2 (Ab-870) Antibody, consider the following methodological recommendations:

• Storage conditions: Store the antibody at 4°C for short-term use (stable for 6 months). For long-term storage, maintain at -20°C and avoid repeated freeze-thaw cycles .

• Buffer composition: Most preparations are supplied at 1.0mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺) at pH 7.4 .

• Western blot optimization:

  • Use freshly prepared lysates when possible

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation state

  • For cell stimulation experiments, utilize PMA (50 ng/mL) and ionomycin (1 μg/mL) for 3 hours to enhance phosphorylation signal

• Positive controls: Consider using cell lines with known NFKB2 activation, such as HepG2 cells transfected with expression vectors encoding NFKB2, as demonstrated in research studies .

• Blocking conditions: Standard blocking with 5% BSA in TBST is typically effective, but optimization may be required depending on your specific experimental system.

How can I validate the specificity of NFKB2 (Ab-870) Antibody in my experiments?

To validate antibody specificity, implement these strategic approaches:

• Phosphorylation-specific controls: Compare samples treated with and without phosphatase to confirm phospho-specificity .

• Peptide competition assay: Pre-incubate the antibody with the phosphorylated peptide used as the immunogen to block specific binding. This approach is validated by the antibody production method where "the antibody against non-phospho peptide was removed by chromatography using corresponding non-phospho peptide" .

• Genetic validation: Use cells with NFKB2 knockdown or knockout as negative controls.

• Stimulation experiments: Treat cells with known activators of the non-canonical NF-κB pathway and observe increased signal .

• Mutation studies: Express wild-type versus mutated NFKB2 (particularly at Ser870) to demonstrate specificity, as demonstrated in research where "HepG2 cells were transfected with expression vectors encoding coding sequence of wild-type NFKB2 gene (pcDNA-NFKB2), or c.1831C > T mutated variant (pcDNA-NFKB2 MUT)" .

How can NFKB2 (Ab-870) Antibody be used to investigate the non-canonical NF-κB signaling pathway?

The NFKB2 (Ab-870) Antibody is a powerful tool for investigating the non-canonical NF-κB pathway through multiple experimental approaches:

• Pathway activation dynamics: Monitor phosphorylation at Ser870 as a marker of pathway activation following stimulation with relevant ligands like lymphotoxin β or CD40L.

• Processing analysis: Examine the correlation between Ser870 phosphorylation and p100 processing to p52, a critical step in non-canonical NF-κB signaling. Research has shown that "impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of IκBδ inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity" .

• Transcriptional regulation: Combine with RT-qPCR analysis of downstream target genes such as CXCL13, CCL19, and MADCAM1 to correlate phosphorylation status with transcriptional outcomes. Published research protocols used specific primers:

  • CXCL13: For 5′-GGACCCTCAAGCTGAATGGA-3′ and Rev 5′-AGCTTGAGTTTGCCCCATCA-3′

  • CCL19: For 5′-GGTGCCTGCTGTAGTGTTCA-3′ and Rev 5′-GCAGTCTCTGGATGATGCGT-3′

  • MADCAM1: For 5′-GTGCTGTTCAGGGTGACAGA-3′ and Rev 5′-GTGCAGGACGGGGATGG-3′

• Subcellular localization: Combine with nuclear/cytoplasmic fractionation to track the movement of phosphorylated NFKB2 between cellular compartments during signaling.

• Crosstalk analysis: Investigate interactions between canonical and non-canonical NF-κB pathways by dual staining with antibodies against components of both pathways.

What methodological approaches can help differentiate between native p100 and processed p52 forms when using NFKB2 antibodies?

Distinguishing between p100 and p52 requires careful experimental design:

• Gel resolution optimization: Use 8-10% SDS-PAGE gels with extended running times to effectively separate the 100 kDa (p100) and 52 kDa (p52) bands.

• Antibody selection strategy: Combine phospho-specific antibodies like NFKB2 (Ab-870) with antibodies targeting different domains:

  • N-terminal antibodies (e.g., AA 1-340) will detect both p100 and p52

  • C-terminal antibodies will detect only p100, not p52

• Immunoblotting techniques: As demonstrated in research studies, "Immunoblot of whole-cell lysates from PBMC isolated from individuals with c.1831C > T mutation (I.1, I.2) and wild-type controls (C1, C2, C3)" can effectively show differences in p100/p52 ratios between experimental and control samples .

• Molecular weight markers: Always include precise molecular weight markers in the 50-120 kDa range to accurately identify each form. The expected molecular weight for p100 is approximately 110 kDa .

• Stimulation experiments: Compare unstimulated versus stimulated conditions to observe dynamic changes in p100/p52 ratios. Research has shown that stimulation with "phorbol 12-myristate 13-acetate (PMA, 50 ng/mL) and ionomycin (1 μg/mL)" for 3 hours can induce processing .

How can NFKB2 (Ab-870) Antibody contribute to studying NFKB2-related immune deficiencies?

NFKB2 (Ab-870) Antibody is particularly valuable for investigating immune deficiencies associated with NFKB2 mutations:

• Mutation impact assessment: Use the antibody to assess how specific mutations affect Ser870 phosphorylation status. Recent research identified "a novel missive heterozygous variant (c.2602T>A:p.Y868N) of NFKB2 in all patients and not in healthy relatives" associated with common variable immune deficiency (CVID) .

• Processing defect characterization: Determine whether mutations alter p100 processing to p52 by comparing phosphorylation patterns and protein ratios between patient and control samples.

• Functional consequences: Correlate phosphorylation defects with downstream functional consequences. Research has demonstrated that "impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of IκBδ inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity" .

• Diagnostic potential: Establish phosphorylation profiles that might serve as diagnostic markers for specific NFKB2-related disorders.

• Therapeutic target identification: Identify potential intervention points in the pathway that might be therapeutically targetable.

What are the key methodological considerations when analyzing NFKB2 phosphorylation in patient samples?

When working with clinical samples to analyze NFKB2 phosphorylation:

• Sample preservation: Immediately process samples and include phosphatase inhibitors to prevent ex vivo dephosphorylation.

• Control selection: Include appropriate age and sex-matched controls. In research studies, comparisons were made between "individuals with c.1831C > T mutation (I.1, I.2) and wild-type controls (C1, C2, C3)" .

• Cell type considerations: Different immune cell populations may exhibit distinct NFKB2 phosphorylation patterns. Consider isolating specific cell subsets for more precise analysis.

• Functional correlation: Combine phosphorylation analysis with functional assays, such as:

  • Immunophenotyping (research has shown "expansion of lymphocyte B subpopulations with concomitant reduction of plasmablasts" in patients)

  • Immunoglobulin level measurement (patients exhibited "low levels of IgG")

  • Gene expression analysis of NFKB2 target genes using RT-PCR

• Viral susceptibility assessment: Recent research has shown that "NFKB2 alleles that are IκBδ GOF and p52 LOF can underlie CVID and drive the production of autoantibodies neutralizing type I IFNs, thereby predisposing to severe viral diseases" . Consider evaluating anti-interferon autoantibodies in patients with NFKB2 mutations.

What are common technical challenges when using NFKB2 (Ab-870) Antibody and how can they be addressed?

Researchers frequently encounter these challenges when working with phospho-specific antibodies like NFKB2 (Ab-870):

• Weak or absent signal:

  • Solution: Ensure the use of fresh phosphatase inhibitors in lysis buffers

  • Optimize antibody concentration (recommended 1:500-1:1000 for WB)

  • Consider signal amplification systems

  • Verify the stimulation protocol activates the non-canonical NF-κB pathway

• High background:

  • Solution: Increase blocking time/concentration

  • Try alternative blocking agents (milk vs. BSA)

  • Increase washing duration and number of washes

  • Reduce antibody concentration

• Non-specific bands:

  • Solution: Use more stringent washing conditions

  • Increase antibody specificity with longer primary antibody incubation at 4°C

  • Consider pre-adsorption with non-phosphorylated peptide

• Inconsistent results between experiments:

  • Solution: Standardize lysate preparation procedures

  • Use consistent positive controls across experiments

  • Normalize loading with appropriate housekeeping proteins (β-actin has been successfully used in published studies)

• Discrepancies between phosphorylation state and functional outcomes:

  • Solution: Consider the timing of sampling relative to stimulation

  • Evaluate additional phosphorylation sites (e.g., Ser866)

  • Examine total NFKB2 levels alongside phosphorylated forms

How can phosphorylation at Ser870 be correctly interpreted in the context of NFKB2 function?

Interpreting Ser870 phosphorylation requires nuanced analysis:

• Pathway context: Ser870 phosphorylation should be considered alongside Ser866 phosphorylation, as dual phosphorylation at Ser866/870 is required for efficient processing of p100 to p52 .

• Temporal dynamics: The kinetics of phosphorylation may vary depending on the stimulus and cell type, necessitating time-course experiments.

• Subcellular localization: Phosphorylated NFKB2 may have different functional consequences depending on its cellular location (cytoplasmic vs. nuclear).

• Correlation with processing: Always assess both phosphorylation and processing (p100 to p52 conversion) to establish a functional relationship.

• Genetic background consideration: Interpret phosphorylation in the context of genetic variations. Research has shown that "NFKB2 haplodeficiency caused by c.1831C > T nonsense mutation is asymptomatic, possibly due to the compensatory mechanisms and allele redundancy" .