RELB Recombinant Monoclonal Antibody

What is RELB and what is its significance in molecular research?

RELB is a transcription factor belonging to the NF-κB/Rel family with a molecular weight of approximately 70 kDa . It plays a critical role in the alternative (non-canonical) NF-κB signaling pathway, which regulates diverse biological processes including lymphoid organ development, B-cell maturation, and immune responses. RELB forms heterodimers with other NF-κB proteins, particularly p52, to regulate transcription of target genes.

Unlike the classical NF-κB pathway components, RELB is involved in more specialized immune functions and has gained significance in studies of lymphoid malignancies, immune disorders, and inflammatory conditions. The accurate detection and quantification of RELB is crucial for understanding its role in normal and pathological conditions, making anti-RELB antibodies indispensable research tools.

What applications are suitable for RELB monoclonal antibodies?

RELB monoclonal antibodies can be utilized in multiple research applications with specific protocols optimized for each technique:

For Western blotting applications, RELB is typically detected as a band at approximately 70 kDa . For optimal results in immunohistochemistry, overnight incubation at 4°C is recommended with appropriate counterstaining using hematoxylin .

What sample types have been validated for RELB antibody detection?

RELB antibodies have been validated on several biological samples:

• Human lymphoma tissue sections, where RELB localizes primarily to the cytoplasm of epithelial cells

• Burkitt's lymphoma cell lines including Daudi and Raji, which express detectable levels of RELB protein

• Various human, mouse, rat, and monkey samples as indicated by the species cross-reactivity data

When working with new sample types, preliminary validation experiments are essential to confirm antibody specificity and optimize detection conditions.

What are the recommended storage and handling procedures for RELB antibodies?

To maintain antibody integrity and performance, follow these guidelines:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles which can compromise antibody function

• For long-term storage: Store at -20°C to -70°C (up to 12 months from date of receipt)

• For short-term storage: After reconstitution, store at 2-8°C under sterile conditions for up to one month

• For extended storage after reconstitution: Store at -20°C to -70°C under sterile conditions for up to 6 months

Always aliquot antibodies upon first thaw to minimize freeze-thaw cycles, and centrifuge briefly before opening vials to collect all material at the bottom of the tube.

How can I validate the specificity of RELB antibodies in my experimental system?

Validation of antibody specificity is crucial for reliable research results. For RELB antibodies, implement these validation approaches:

• Positive control selection: Use cell lines with known RELB expression such as Daudi or Raji human Burkitt's lymphoma cell lines, which have been demonstrated to express detectable levels of RELB .

• Multiple detection methods: Confirm RELB detection using complementary techniques. For example, if your primary technique is Western blotting, validate findings using immunocytochemistry or immunoprecipitation to confirm target specificity.

• siRNA knockdown or CRISPR knockout: Generate RELB-depleted samples through genetic manipulation. A specific antibody should show reduced or absent signal in knockdown/knockout samples compared to wild-type controls.

• Peptide competition assay: Pre-incubate the antibody with a synthetic peptide corresponding to the immunogen. If the antibody is specific, pre-incubation should block detection in subsequent assays.

• Multiple antibody approach: Use different antibodies targeting distinct epitopes of RELB. Concordant results from multiple antibodies increase confidence in specificity.

When performing Western blot validation, the RELB protein should appear as a specific band at approximately 70 kDa, as demonstrated in lysates from Daudi and Raji cell lines .

What analytical techniques are most appropriate for characterizing RELB using monoclonal antibodies?

Several analytical techniques can be employed to characterize RELB using monoclonal antibodies, each offering distinct advantages:

• Chromatographic methods: Reversed-phase liquid chromatography (RPLC) coupled with mass spectrometry (RPLC-MS) enables separation of antibody subdomains and detection of post-translational modifications that may affect RELB binding . This approach allows both qualitative and quantitative assessment of antibody heterogeneity.

• Electrophoretic techniques: Capillary electrophoresis methods, including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and capillary isoelectric focusing (cIEF), offer high resolving power for separating RELB antibodies and assessing their heterogeneity based on charge and size .

• Spectroscopic approaches: Nuclear Magnetic Resonance (NMR) spectroscopy, particularly 2D NMR, provides detailed structural information about RELB antibodies at the atomic level, generating molecular fingerprints that reveal high-order structural characteristics .

• Surface Plasmon Resonance (SPR): This technique allows real-time measurement of binding kinetics between RELB antibodies and their target antigen, providing crucial data on affinity, avidity, and immunoreactivity .

• Multi-angle light scattering (MALS): When coupled with RP-UPLC, MALS enables characterization of intact RELB antibodies and their fragments, calculating molecular weight for each chromatographic peak to identify monomeric variants and potential degradation products .

For comprehensive characterization, a combination of these techniques is recommended to assess different aspects of RELB antibody quality and function.

How do post-translational modifications affect RELB antibody binding and detection?

Post-translational modifications (PTMs) can significantly impact RELB antibody binding through several mechanisms:

• Altered epitope accessibility: PTMs such as phosphorylation, glycosylation, or ubiquitination may modify the three-dimensional structure of RELB, potentially masking or exposing epitopes recognized by specific antibodies.

• Charge distribution changes: Modifications that alter the charge of RELB (e.g., phosphorylation, acetylation, deamidation) can affect its electrophoretic mobility and chromatographic behavior. Ion-exchange chromatography (IEX) is particularly useful for detecting such charge variants .

• Conformational changes: PTMs can induce structural changes in RELB that may impact antibody recognition, especially for antibodies targeting conformational epitopes rather than linear sequences.

• Variable detection in different cellular contexts: Different cell types or conditions may produce RELB with distinct PTM patterns, leading to variable antibody reactivity across samples. For example, the phosphorylation status of RELB often differs between resting and activated immune cells.

To address the heterogeneity arising from PTMs, analytical techniques such as reversed-phase LC-MS (RPLC-MS) can be employed to separate RELB with various modifications, including pyroglutamic acid formation, isomerization, deamidation, and oxidation . This approach enables both qualitative and quantitative assessment of RELB heterogeneity.

When investigating PTMs of RELB, consider implementing peptide mapping strategies to identify modification sites and quantify modification levels at each site. This information can guide selection of appropriate antibodies that are either sensitive or insensitive to specific modifications, depending on research objectives.

What are the optimal conditions for using RELB antibodies in different experimental techniques?

The optimal conditions for using RELB antibodies vary by application:

Western Blotting:

• Recommended dilution: 1:1000

• Sample preparation: Reducing conditions show clear detection of RELB at 70 kDa

• Buffer system: Use Immunoblot Buffer Group 4 for optimal results

• Secondary antibody: HRP-conjugated anti-mouse/rabbit IgG depending on primary antibody source

• Detection: Enhanced chemiluminescence (ECL) for sensitive detection

Immunohistochemistry (Paraffin Sections):

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Antibody concentration: 8 μg/mL

• Incubation: Overnight at 4°C for optimal binding

• Detection system: HRP-DAB (brown) with hematoxylin counterstain (blue)

• Controls: Include lymphoma tissue as positive control where RELB localizes to cytoplasm in epithelial cells

Immunofluorescence/Immunocytochemistry:

• Cell fixation: Immersion fixation method

• Antibody concentration: 25 μg/mL

• Incubation time: 3 hours at room temperature

• Secondary antibody: Fluorochrome-conjugated (e.g., NorthernLights 557)

• Counterstain: DAPI for nuclear visualization

• Expected localization: Primarily cytoplasmic in Burkitt's lymphoma cells

Immunoprecipitation:

• Recommended dilution: 1:100

• Lysate preparation: Use RIPA buffer with protease and phosphatase inhibitors

• Protein A/G beads: Pre-clear lysate before adding antibody

• Incubation: Overnight at 4°C on a rotator

• Washes: Minimum of 3-5 washes with appropriate buffer to reduce background

Each technique requires optimization based on specific experimental conditions and sample types. Preliminary titration experiments are recommended to determine optimal antibody concentrations for your specific application.

How can I troubleshoot inconsistent results when using RELB antibodies in Western blotting?

When encountering inconsistent results with RELB antibodies in Western blotting, follow this systematic troubleshooting approach:

• Sample preparation issues:

  • Ensure complete cell lysis and protein extraction

  • Add fresh protease inhibitors to prevent RELB degradation

  • Confirm protein concentration using reliable methods (BCA or Bradford assay)

  • Validate sample integrity by blotting for a stable housekeeping protein

• Gel electrophoresis parameters:

  • Use appropriate acrylamide percentage (8-10% for 70 kDa RELB)

  • Ensure complete transfer to membrane by staining the gel post-transfer

  • Verify equal loading using total protein stains (Ponceau S) or housekeeping controls

• Antibody-specific factors:

  • Confirm antibody activity using positive controls (Daudi or Raji cell lysates)

  • Test multiple RELB antibodies targeting different epitopes

  • Optimize antibody concentration (starting with 1:1000 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

• Detection system optimization:

  • Ensure compatible secondary antibody (HRP-conjugated anti-mouse IgG for MAB2698)

  • Use appropriate buffer system (Immunoblot Buffer Group 4)

  • Optimize exposure time to prevent over or under-exposure

  • Consider enhanced sensitivity detection methods for low-abundance samples

• Technical considerations:

  • Always run the Western blot under reducing conditions

  • Ensure membranes are properly blocked to reduce background

  • Optimize washing steps (duration, volume, buffer composition)

  • Consider fresh preparation of all working solutions

If bands appear at unexpected molecular weights, evaluate potential post-translational modifications, proteolytic processing, or isoform expression. Cross-validation with orthogonal techniques (immunoprecipitation, immunocytochemistry) can help confirm the specificity of detected bands.

What considerations should be made when designing experiments to study RELB in the context of NF-κB signaling?

When designing experiments to investigate RELB in NF-κB signaling, consider these important factors:

• Pathway activation specificity:

  • RELB primarily functions in the non-canonical NF-κB pathway

  • Select appropriate stimuli that specifically activate this pathway (e.g., CD40L, BAFF, lymphotoxin-β)

  • Include controls for canonical pathway activation (e.g., TNF-α, IL-1β) for comparison

• Temporal dynamics:

  • Non-canonical pathway activation is typically slower than canonical signaling

  • Design time-course experiments that capture both early (1-6 hours) and late (12-48 hours) events

  • Monitor RELB nuclear translocation kinetics, which differ from classical NF-κB components

• Protein interaction analysis:

  • Investigate RELB dimerization partners (primarily p52)

  • Use co-immunoprecipitation with RELB antibodies (1:100 dilution) to pull down interaction partners

  • Consider chromatin immunoprecipitation (ChIP) to identify RELB-bound genomic regions

• Subcellular localization:

  • Monitor RELB translocation between cytoplasm and nucleus

  • Perform subcellular fractionation followed by Western blotting

  • Use immunocytochemistry with RELB antibodies (25 μg/mL) to visualize localization patterns

• Functional readouts:

  • Assess transcriptional activity of RELB-dependent genes

  • Measure phenotypic outcomes relevant to the cell type and context

  • Consider pathway crosstalk with other signaling systems

• Model system selection:

  • Choose appropriate cell types where RELB signaling is biologically relevant

  • Consider lymphoid cell lines like Daudi and Raji that express detectable RELB levels

  • When using tissue samples, lymphoma tissues have been validated for RELB detection

When interpreting results, remember that RELB functions distinctly from other NF-κB family members and exhibits cell type-specific effects. Parallel assessment of multiple NF-κB components provides a more comprehensive understanding of pathway dynamics.

How can I quantitatively assess RELB expression levels across different experimental conditions?

Accurate quantitative assessment of RELB expression requires rigorous methodological approaches:

• Western blot quantification:

  • Use gradient gels (4-15%) for optimal separation of the 70 kDa RELB protein

  • Include recombinant RELB standards at known concentrations for standard curve generation

  • Capture images using a digital imaging system with linear detection range

  • Analyze band intensity using software (ImageJ, Image Lab) with background subtraction

  • Normalize RELB signal to housekeeping proteins or total protein stains

  • Run technical triplicates to calculate coefficient of variation (CV < 15% is acceptable)

• Enzyme-linked immunosorbent assay (ELISA):

  • Develop a sandwich ELISA using capture and detection antibodies against different RELB epitopes

  • Establish a standard curve using recombinant RELB protein

  • Validate assay parameters (sensitivity, specificity, precision, accuracy)

  • Calculate intra-assay and inter-assay variability to ensure reproducibility

  • Determine the linear range of detection for your sample types

• Mass spectrometry-based quantification:

  • Implement targeted proteomics approaches such as selected reaction monitoring (SRM)

  • Use stable isotope-labeled peptide standards for absolute quantification

  • Select proteotypic peptides specific to RELB

  • Employ RPLC for separation of peptides before MS analysis

  • Perform statistical validation of quantitative results

• Flow cytometry:

  • Optimize cell fixation and permeabilization for intracellular RELB detection

  • Include isotype controls and fluorescence-minus-one (FMO) controls

  • Calibrate using beads with known antibody binding capacity

  • Report results as molecules of equivalent soluble fluorochrome (MESF)

  • Consider phospho-specific antibodies for activation state assessment

• Real-time quantitative PCR (RT-qPCR):

  • Design primers specific to RELB mRNA

  • Validate primer efficiency (90-110%) and specificity

  • Use multiple reference genes for normalization

  • Apply the 2^(-ΔΔCt) method for relative quantification

  • Correlate mRNA levels with protein expression to assess post-transcriptional regulation

When comparing RELB levels across experimental conditions, ensure consistent sample preparation, equal loading, and appropriate statistical analysis to detect significant differences.

What are the advantages and limitations of recombinant monoclonal antibodies versus conventional monoclonal antibodies for RELB research?

Understanding the differences between recombinant and conventional monoclonal antibodies is crucial for selecting the appropriate tool for RELB research:

Advantages of Recombinant Monoclonal Antibodies:

• Enhanced reproducibility: Recombinant antibodies are produced from sequenced DNA in defined expression systems, eliminating batch-to-batch variability inherent in hybridoma-derived antibodies .

• Improved specificity: The defined sequence and production process allow for precise epitope targeting and reduced cross-reactivity with similar proteins.

• Engineered modifications: Recombinant technology enables specific modifications such as humanization, isotype switching, or affinity maturation to enhance antibody performance.

• Ethical considerations: Production doesn't require animals after initial sequence determination, addressing ethical concerns related to hybridoma generation and maintenance.

• Consistency in post-translational modifications: When produced in controlled expression systems, recombinant antibodies show more consistent glycosylation patterns and other PTMs .

Limitations of Recombinant Monoclonal Antibodies:

• Higher production costs: Recombinant production systems typically require more sophisticated infrastructure and expertise, increasing costs.

• Potential epitope constraints: Some recombinant antibodies may recognize linear epitopes more efficiently than conformational epitopes, depending on design strategy.

• Expression system limitations: The choice of expression system can introduce non-native modifications that might affect antibody function.

• Technical expertise requirements: Working with recombinant antibodies often requires specialized knowledge in molecular biology and protein engineering.

Comparative Analysis for RELB Research Applications:

Key considerations for successful RELB antibody implementation in research

When implementing RELB antibodies in research, several critical factors must be considered to ensure reliable and reproducible results:

• Experimental design should include appropriate positive controls (such as Daudi or Raji cell lysates) and negative controls to validate antibody specificity .

• Optimization of protocols for specific applications is essential, with particular attention to antibody dilution, incubation conditions, and detection methods .

• Understanding the molecular characteristics of RELB, including its molecular weight (70 kDa), cellular localization (primarily cytoplasmic in many cell types), and participation in the non-canonical NF-κB pathway, is fundamental to correct interpretation of results .

• Comprehensive validation strategies, including multiple detection methods and analytical techniques, provide stronger evidence for observed RELB expression patterns and functional roles.

• Consideration of post-translational modifications and their impact on RELB detection can help explain variability across different experimental conditions or sample types .