IKBKG (Ab-85) Antibody

What is the IKBKG (Ab-85) Antibody and what does it target?

The IKBKG (Ab-85) Antibody is a rabbit polyclonal antibody that specifically recognizes the serine 85 phosphorylation site on the IKK gamma/NEMO protein (also known as Inhibitor of kappa Light Polypeptide Gene Enhancer in B-Cells, Kinase gamma). This antibody is designed to detect the phosphorylated form of IKBKG at serine 85 within the amino acid sequence context Q-A-S(p)-Q-R. The antibody is generated using a synthesized non-phosphopeptide derived from human IKK-gamma as the immunogen and is affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography .

What applications can the IKBKG (Ab-85) Antibody be used for?

The IKBKG (Ab-85) Antibody has been validated for several experimental applications including:

• Western Blotting (WB): For detecting the protein in cell lysates and tissue samples

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection

• Immunofluorescence (IF): For visualizing protein localization in cells or tissues

The recommended dilutions for optimal results vary by application: 1:500-1:3000 for Western blotting, 1:2000-1:10000 for ELISA, and 1:100-1:500 for immunofluorescence .

What species does the IKBKG (Ab-85) Antibody react with?

The IKBKG (Ab-85) Antibody has been validated to cross-react with both human and mouse IKBKG proteins. This makes it suitable for comparative studies between these two species, particularly in research focusing on conserved signaling pathways or disease models .

How should I design a Western blot experiment using IKBKG (Ab-85) Antibody?

When designing a Western blot experiment with the IKBKG (Ab-85) Antibody, consider the following methodology:

• Sample preparation: Extract proteins using a lysis buffer containing phosphatase inhibitors to preserve the phosphorylation state of Ser85.

• Protein separation: Use 10-12% SDS-PAGE gels for optimal resolution of IKBKG (approximately 48 kDa).

• Transfer conditions: Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer.

• Blocking: Block the membrane with 5% BSA in TBST (not milk, as it contains phosphatases).

• Primary antibody incubation: Dilute IKBKG (Ab-85) Antibody at 1:1000 in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use an anti-rabbit HRP-conjugated secondary antibody (1:5000).

• Controls: Include positive controls (cells treated with stimuli known to induce IKBKG phosphorylation) and negative controls (phosphatase-treated samples).

This method allows for specific detection of the phosphorylated form of IKBKG at Ser85, which is important for monitoring NF-κB pathway activation .

What are effective immunofluorescence protocols for IKBKG (Ab-85) Antibody?

For optimal immunofluorescence results with the IKBKG (Ab-85) Antibody:

• Cell preparation: Culture cells on coverslips and fix with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: Use 0.2% Triton X-100 in PBS for 10 minutes to allow antibody access to intracellular antigens.

• Blocking: Block with 5% normal serum in PBS with 0.1% Tween-20 for 1 hour.

• Primary antibody: Dilute IKBKG (Ab-85) Antibody at 1:200 in blocking solution and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated anti-rabbit secondary antibody (1:500) for 1 hour at room temperature.

• Counterstaining: DAPI (1:1000) for nuclear visualization.

• Mounting: Mount using anti-fade mounting medium.

This protocol allows visualization of phosphorylated IKBKG localization, which is often observed in both cytoplasmic and nuclear compartments following pathway activation .

How can IKBKG (Ab-85) Antibody be used to study NF-κB signaling dynamics?

The IKBKG (Ab-85) Antibody is an essential tool for studying the temporal dynamics of NF-κB pathway activation through the following methodological approaches:

• Time-course experiments: Treat cells with relevant stimuli (TNF-α, IL-1β, LPS) at different time points and monitor Ser85 phosphorylation status.

• Subcellular fractionation: Combine with cellular fractionation to track the movement of phosphorylated IKBKG between cytoplasmic and nuclear compartments.

• Co-immunoprecipitation: Use the antibody to pull down pSer85-IKBKG and identify interaction partners during signaling.

• Phosphorylation kinetics: Compare phosphorylation at Ser85 with other phosphorylation sites (such as Ser31) to determine the sequence of phosphorylation events.

• Inhibitor studies: Apply specific kinase inhibitors to determine which upstream signals regulate Ser85 phosphorylation.

This approach provides insights into the temporal regulation of NF-κB signaling, which is critical for understanding inflammatory and immune responses .

What is the role of IKBKG in polyubiquitination-mediated signaling and how can the antibody help study this process?

The IKBKG (Ab-85) Antibody can be instrumental in investigating polyubiquitination-mediated signaling through these research approaches:

• Sequential immunoprecipitation: First pull down IKBKG using a total IKBKG antibody, then probe for ubiquitin chains using specific antibodies against K63-linked or linear polyubiquitin.

• Reverse co-IP experiments: Immunoprecipitate with ubiquitin antibodies and then probe for phospho-Ser85 IKBKG.

• Correlation analysis: Compare levels of phospho-Ser85 IKBKG with polyubiquitination status in response to different stimuli.

• Ubiquitin mutant expression: Express ubiquitin mutants (K63R, K27R) and examine effects on Ser85 phosphorylation using the antibody.

• Deubiquitinase inhibitor studies: Treat cells with DUB inhibitors and monitor changes in Ser85 phosphorylation.

These approaches help elucidate how different types of polyubiquitin chains (K63-linked, linear, K27-linked) interact with IKBKG and influence its phosphorylation and subsequent NF-κB activation .

What are common issues when using IKBKG (Ab-85) Antibody in Western blotting and how can they be resolved?

When working with IKBKG (Ab-85) Antibody in Western blotting, researchers may encounter these challenges and solutions:

• High background:

  • Solution: Increase blocking time to 2 hours, use 0.05% Tween-20 in wash buffers, and optimize antibody dilution (try 1:2000).

  • Methodological approach: Perform a titration experiment with different antibody concentrations.

• Weak or no signal:

  • Solution: Confirm phosphorylation status with appropriate positive controls (TNF-α or IL-1β stimulated cells).

  • Methodological approach: Include phosphatase inhibitors in all buffers and avoid freeze-thaw cycles of samples.

• Multiple bands:

  • Solution: Use alternative blocking agents like 5% BSA instead of milk.

  • Methodological approach: Perform peptide competition assays to confirm specificity.

• Inconsistent results:

  • Solution: Standardize lysate preparation and protein loading.

  • Methodological approach: Use total IKBKG antibody in parallel to normalize phospho-specific signals.

These troubleshooting methods help ensure reliable and reproducible detection of phosphorylated IKBKG in Western blotting applications .

How can researchers distinguish between IKBKG gene and its pseudogene (IKBKGP) in molecular studies?

Distinguishing between the IKBKG gene and its highly homologous pseudogene IKBKGP requires specific methodological approaches:

• Long-range PCR strategy:

  • Amplify exons 2-10 in IKBKG using primers that target unique regions not present in the pseudogene.

  • Follow with nested PCR for specific exons (particularly exon 8).

  • Use primers: forward 5′-TCGTCAGCAGGCAATAGTTAGTTGGTTGA-3′ and reverse 5′-TATGCCAAAGATACGCACGACTAATGCAC-3′ for initial long-range PCR.

• MLPA analysis:

  • Use SALSA MLPA probemix P073-A1 specifically designed to evaluate both IKBKG and IKBKGP.

  • Analyze relative copy numbers after normalization against control samples.

• Deep sequencing approach:

  • Perform NGS after long-range PCR to detect low-level mosaicism of single nucleotide variants.

  • Compare with genomic sequences from control samples.

• Nested PCR for mosaic deletions:

  • Use a nested PCR approach with two rounds of amplification.

  • Dilute first-round products 1:100 with TE buffer before the second amplification.

These techniques are crucial for accurate genetic analysis in research settings, particularly when studying conditions like Incontinentia pigmenti where IKBKG mutations play a causal role .

What is the functional significance of IKBKG Ser85 phosphorylation in different signaling pathways?

The phosphorylation of IKBKG at Ser85 has significant implications for multiple signaling pathways:

• NF-κB pathway activation:

  • Ser85 phosphorylation facilitates the formation of the active IKK complex.

  • This leads to phosphorylation of IκB inhibitors, their degradation, and subsequent NF-κB nuclear translocation.

  • The phosphorylation state can be monitored as an indicator of pathway activation intensity and duration.

• Antiviral innate immune responses:

  • Phosphorylated IKBKG is essential for viral activation of IRF3.

  • It plays a crucial role in TLR3- and IFIH1-mediated antiviral responses.

  • These functions require coordination with K27-linked polyubiquitination.

• Cytokine-mediated signaling:

  • Ser85 phosphorylation status affects NF-κB-mediated protection from cytokine toxicity.

  • It influences pro-inflammatory cytokine production in various cell types.

• Pathogen recognition:

  • The phosphorylation plays a role in responses to microbial infection.

  • It can mediate HTLV-1 Tax oncoprotein activation of NF-κB.

Understanding these pathways through phospho-specific antibody detection provides insights into normal immune function and disease mechanisms .

How do mutations in IKBKG relate to disease, and how can the antibody be used to study these mechanisms?

IKBKG mutations are associated with several human diseases, and the IKBKG (Ab-85) Antibody can be instrumental in studying their mechanisms:

• Incontinentia pigmenti (IP):

  • A rare X-linked disorder affecting skin and other ectodermal tissues

  • The antibody can be used to compare phosphorylation patterns between wild-type and mutant IKBKG in patient-derived cells.

  • Research methodology: Establish patient-derived cell lines and examine Ser85 phosphorylation in response to pathway stimulation.

• Low-level mosaicism detection:

  • Some patients carry IKBKG mutations in only a subset of cells.

  • Combine immunostaining with the phospho-specific antibody and genetic analysis to identify mosaic expression patterns.

  • Research methodology: Use the antibody in single-cell analysis approaches to detect cellular heterogeneity.

• Functional consequences of different mutations:

  • The antibody can help characterize how specific mutations (like the nonsense mutation c.924C>G; p.Tyr308*) affect Ser85 phosphorylation.

  • Research methodology: Express IKBKG mutants in cell models and compare phosphorylation efficiency using the antibody.

• X-chromosome inactivation studies:

  • In female patients with heterozygous mutations, X-inactivation patterns influence disease severity.

  • The antibody can help visualize cellular mosaicism resulting from random X-inactivation.

  • Research methodology: Combine HUMARA assay results with immunofluorescence using the phospho-specific antibody.

These approaches help translate genetic findings to functional outcomes, providing mechanistic insights into disease pathogenesis .