IKBKB Antibody

What is IKBKB and why is it significant in cellular signaling research?

IKBKB, also known as IKK-beta, IKK2, or IKKB, is a key kinase that phosphorylates the inhibitor in the inhibitor/NF-kappa-B complex, causing dissociation of the inhibitor and subsequent activation of NF-kappa-B . The protein exists as part of a larger protein complex that plays a crucial role in immune response regulation, cell survival, and inflammation pathways. IKBKB is a serine/threonine protein kinase with a molecular weight of approximately 87 kDa . As a central regulator of NF-κB activation, IKBKB is implicated in various disease processes, making it an important target for both basic research and therapeutic development studies.

How do monoclonal and polyclonal IKBKB antibodies differ in research applications?

Monoclonal IKBKB antibodies, such as those produced in mouse hosts, recognize a single epitope of the IKBKB protein and offer high specificity for targeted applications . For instance, mouse-derived monoclonal antibodies are commonly used in ELISA and IHC applications with optimal dilutions of 1/10000 for ELISA and 1/200-1/1000 for IHC .

Polyclonal IKBKB antibodies, typically produced in rabbits, recognize multiple epitopes on the IKBKB protein, providing higher sensitivity but potentially lower specificity than monoclonal antibodies . They are versatile across multiple applications including Western blotting (recommended dilutions 1:300-1:1000), immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate), and immunohistochemistry (1:50-1:500) . The broader epitope recognition of polyclonal antibodies makes them particularly valuable for detecting proteins in denatured conditions or when protein expression levels are low.

What are the common aliases and molecular characteristics of IKBKB that researchers should be aware of?

Researchers should recognize multiple aliases when searching literature or antibody products:

Regarding molecular characteristics, IKBKB has a calculated molecular weight ranging from 81-87 kDa, though observed weights in experimental conditions may include bands at 80 kDa, 86 kDa, and 87 kDa . Some antibodies may also detect a smaller fragment at approximately 29 kDa, representing an alternative splice variant or proteolytic product .

What are the optimal applications for IKBKB antibodies in molecular research?

IKBKB antibodies have demonstrated utility across numerous experimental applications:

When selecting an application, researchers should consider the specific experimental question, available samples, and whether native protein conformation is required for detection.

How should Western blotting protocols be optimized when using IKBKB antibodies?

For optimal Western blot results with IKBKB antibodies, researchers should consider the following protocol modifications:

• Sample preparation: IKBKB has been successfully detected in various cell types including Jurkat, K-562, and HepG2 cells . Lysis buffers containing phosphatase inhibitors are essential as IKBKB is a phosphoprotein.

• Gel electrophoresis: Use 8-10% SDS-PAGE gels for optimal resolution of the 87 kDa IKBKB protein.

• Transfer conditions: Extended transfer times (1-2 hours) or lower current overnight transfers may improve transfer efficiency for this higher molecular weight protein.

• Blocking: 5% non-fat dry milk or BSA in TBST is typically effective, though specific antibodies may have optimized recommendations.

• Antibody incubation: Primary antibody dilutions ranging from 1:300-1:2000 have been validated, with overnight incubation at 4°C providing optimal signal-to-noise ratios .

• Visualization: Both chemiluminescence and fluorescence-based detection methods are compatible with IKBKB antibodies.

• Controls: Include positive controls such as lysates from Jurkat cells, which are known to express IKBKB at detectable levels .

Researchers should be prepared to observe multiple bands, as IKBKB may present at 80 kDa, 86 kDa, 87 kDa, and potentially a smaller 29 kDa fragment depending on the antibody's epitope recognition and the sample's post-translational modifications .

What are the best practices for immunohistochemistry with IKBKB antibodies?

For successful immunohistochemistry using IKBKB antibodies, researchers should implement these best practices:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) tissues have been successfully used with IKBKB antibodies, particularly in human liver cancer and prostate cancer samples .

• Antigen retrieval: Two effective methods have been documented:

  • TE buffer at pH 9.0 (preferred method)

  • Citrate buffer at pH 6.0 (alternative method)

• Blocking: Use appropriate serum corresponding to the secondary antibody's host species to minimize background staining.

• Primary antibody incubation: Dilutions between 1:50-1:500 have been validated, with overnight incubation at 4°C recommended for optimal staining .

• Detection systems: Both DAB-based and fluorescence-based detection systems are compatible with IKBKB antibodies.

• Counterstaining: Hematoxylin provides good nuclear contrast when using chromogenic detection methods.

• Specificity controls: Include negative controls (primary antibody omission) and, when possible, tissues from IKBKB-knockout models or those treated with IKBKB-specific siRNA.

Why might I observe multiple bands in Western blot when using IKBKB antibodies?

Multiple bands in Western blots using IKBKB antibodies are commonly observed and may result from several biological and technical factors:

• Alternative splice variants: IKBKB has multiple isoforms with calculated molecular weights of approximately 81 kDa (756 amino acids) and 29 kDa (256 amino acids) .

• Post-translational modifications: IKBKB undergoes phosphorylation and other modifications that can alter its migration pattern. This explains why observed molecular weights (80 kDa, 86 kDa, 87 kDa) may differ from calculated values .

• Proteolytic processing: Some IKBKB fragments may result from biological or sample preparation-induced proteolysis.

• Cross-reactivity: Antibodies, particularly polyclonal variants, may recognize structurally similar proteins in the IKK family.

• Sample preparation effects: The mobility of proteins in SDS-PAGE can be affected by buffer components, reducing agents, and heating conditions .

As noted by Elabscience: "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. The common factors include: If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane."

How can I determine the specificity of my IKBKB antibody?

Verifying antibody specificity is crucial for result interpretation. Consider these validation approaches:

• Positive control samples: Use cell lines known to express IKBKB, such as Jurkat, K-562, or HepG2 cells .

• Knock-down/knock-out controls: Compare antibody staining between wild-type samples and those with reduced IKBKB expression via siRNA, shRNA, or CRISPR-Cas9 gene editing.

• Competing peptide assay: Pre-incubate the antibody with the immunogen peptide used to generate it. This should block specific binding and eliminate true IKBKB signal.

• Cross-reference with different antibodies: Use antibodies targeting different IKBKB epitopes and compare staining patterns.

• Immunoprecipitation-mass spectrometry: Perform IP with the IKBKB antibody followed by mass spectrometry analysis to confirm captured proteins.

• Recombinant protein testing: Test antibody detection limits and specificity using purified recombinant IKBKB protein.

What factors affect epitope accessibility in fixed tissues when using IKBKB antibodies?

Several factors influence epitope accessibility in fixed tissues when using IKBKB antibodies for immunohistochemistry:

• Fixation method: Overfixation can mask epitopes through extensive protein cross-linking. IKBKB antibodies have been validated primarily on formalin-fixed tissues .

• Antigen retrieval techniques: Heat-induced epitope retrieval (HIER) using TE buffer at pH 9.0 is recommended for IKBKB detection, with citrate buffer at pH 6.0 as an alternative .

• Antibody epitope location: Epitopes located within the protein's hydrophobic core may be less accessible than surface epitopes. The immunogen sequence can provide insight into potential accessibility challenges.

• Cellular localization: IKBKB has been reported in multiple cellular compartments including cytoplasm, membrane rafts, and nucleus , which may require different permeabilization approaches.

• Protein-protein interactions: IKBKB exists in protein complexes that may mask certain epitopes in its native state.

• Post-translational modifications: Phosphorylation or other modifications may alter epitope recognition, particularly if the antibody was raised against unmodified protein sequences .

How can IKBKB antibodies be used to study the NF-κB signaling pathway dynamics?

IKBKB antibodies enable sophisticated studies of NF-κB signaling dynamics through several advanced applications:

• Phospho-specific detection: Some antibodies specifically recognize phosphorylated forms of IKBKB, allowing researchers to monitor its activation state following various stimuli.

• Kinase activity assays: Immunoprecipitated IKBKB can be used in kinase assays to directly measure enzymatic activity against IκB substrates.

• Co-immunoprecipitation studies: IKBKB antibodies have been validated for CoIP applications, allowing researchers to pull down entire signaling complexes and study protein-protein interactions within the NF-κB pathway .

• Live-cell imaging: Fluorescently tagged antibody fragments or nanobodies against IKBKB can be used to visualize signaling dynamics in living cells.

• Chromatin immunoprecipitation (ChIP): IKBKB antibodies can help identify genomic loci where IKBKB may be recruited as part of transcriptional regulation complexes.

• Proximity ligation assays: These can detect and quantify IKBKB interactions with other pathway components with high spatial resolution.

• Tissue microarray analysis: IKBKB antibodies can be used to analyze pathway activation across multiple patient samples or experimental conditions simultaneously.

What considerations are important when selecting IKBKB antibodies for cross-species studies?

When planning cross-species experiments with IKBKB antibodies, researchers should consider:

• Validated reactivity: Different antibodies show varied species reactivity profiles. Some IKBKB antibodies have been validated for human, mouse, and rat samples , while others may be species-restricted .

• Epitope conservation: Examine the sequence homology between species at the immunogen region. Higher conservation generally predicts better cross-reactivity.

• Application-specific validation: An antibody that works in Western blot across species may not work equally well in IHC or IF applications.

• Isoform specificity: Different species may express different IKBKB isoforms or splice variants that could affect antibody recognition.

• Background concerns: Non-specific binding patterns may differ between species, requiring optimization of blocking conditions and antibody concentrations.

• Positive controls: Include known positive samples from each species being studied to confirm cross-reactivity.

According to available data, antibodies like the polyclonal rabbit anti-IKBKB (E-AB-60595) have demonstrated reactivity with human, mouse, and rat samples across Western blot and immunohistochemistry applications , making them suitable for comparative studies.

How can researchers differentiate between IKBKB isoforms using available antibodies?

Differentiating between IKBKB isoforms requires strategic antibody selection and experimental design:

• Epitope mapping: Select antibodies raised against peptides specific to particular isoforms or regions present/absent in certain splice variants.

• Molecular weight discrimination: IKBKB has reported isoforms with calculated molecular weights of approximately 81 kDa and 29 kDa . Western blotting can separate these based on size.

• Isoform-specific knockdown: Use siRNAs targeting specific isoforms to confirm antibody specificity.

• Subcellular localization: Different isoforms may localize to different cellular compartments (cytoplasm, membrane rafts, nucleus) . Use subcellular fractionation followed by Western blotting or immunofluorescence to distinguish localization patterns.

• Post-translational modification profiling: Isoforms may differ in their modification sites. Antibodies specific to certain phosphorylated, acetylated, or otherwise modified regions can help distinguish isoforms.

• Expression pattern analysis: Different tissues may preferentially express certain isoforms. Comparing antibody reactivity across tissue types can provide insights into isoform distribution.

What are the optimal sample preparation methods for detecting IKBKB in different experimental systems?

Sample preparation methods should be tailored to both the experimental system and the intended application:

For Western blotting:

• Cell lysate preparation: RIPA buffer with protease and phosphatase inhibitors works well for most cell types. Jurkat, K-562, and HepG2 cells have been validated as positive controls .

• Tissue homogenization: Tissues should be homogenized in appropriate buffers at 4°C to prevent protein degradation.

• Protein quantification: Bradford or BCA assays ensure equal loading across samples.

• Denaturation: Samples should be heated at 95°C for 5 minutes in Laemmli buffer with reducing agents.

For immunohistochemistry:

• Fixation: 10% neutral buffered formalin for 24-48 hours is standard.

• Processing and embedding: Standard paraffin embedding protocols.

• Sectioning: 4-6 μm sections are optimal for IKBKB staining.

• Antigen retrieval: TE buffer at pH 9.0 (preferred) or citrate buffer at pH 6.0 (alternative) .

For immunoprecipitation:

• Gentler lysis buffers (e.g., NP-40 or Triton X-100 based) that preserve protein-protein interactions.

• Pre-clearing with protein A/G beads to reduce non-specific binding.

• Antibody amounts between 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate .

How should IKBKB antibodies be stored to maintain optimal reactivity over time?

Proper storage is critical for maintaining antibody performance over time:

• Temperature: Store IKBKB antibodies at -20°C for long-term storage . Avoid freezing and thawing cycles as they can degrade antibody quality.

• Formulation: Most commercial IKBKB antibodies are supplied in:

  • Buffered aqueous solutions containing stabilizers

  • PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Phosphate buffered solution, pH 7.4, containing 0.05% stabilizer and 50% glycerol

• Aliquoting: Divide antibodies into small, single-use aliquots upon receipt to minimize freeze-thaw cycles.

• Working solutions: Keep diluted working solutions at 4°C for short-term use (1-2 weeks).

• Shipping conditions: IKBKB antibodies are typically shipped with ice packs and should be stored immediately at the recommended temperature upon receipt .

• Stability: When properly stored, most IKBKB antibodies remain stable for 12 months from the date of receipt .

• Preservatives: Sodium azide (0.02-0.05%) is commonly included as a preservative and prevents microbial contamination.

What validation data should researchers request when selecting an IKBKB antibody for their specific application?

When selecting an IKBKB antibody, researchers should request comprehensive validation data including:

• Application-specific validation: Evidence that the antibody works in your intended application (WB, IHC, IF, IP, etc.) with representative images.

• Specificity controls: Data showing antibody specificity through knockout/knockdown experiments, blocking peptides, or other validation methods.

• Species reactivity: Experimental validation of reactivity with your species of interest, not just sequence homology predictions.

• Sensitivity information: Limits of detection in relevant applications and sample types.

• Lot-to-lot consistency data: Evidence of reproducibility between different antibody lots.

• Immunogen information: The exact peptide or protein fragment used to generate the antibody, which helps predict epitope accessibility in various applications.

• Recommended protocols: Detailed methods including buffer compositions, incubation times, and troubleshooting suggestions.

• Citation list: Peer-reviewed publications that have successfully used the antibody in relevant applications.

• Cross-reactivity testing: Data showing the antibody's specificity against related proteins in the IKK family.

• Clone information: For monoclonal antibodies, information about the clone and isotype provides insight into potential applications and secondary antibody selection.