IRF2BP1 Antibody, Biotin conjugated

What is IRF2BP1 and why is it significant in molecular research?

IRF2BP1 (Interferon Regulatory Factor 2 Binding Protein 1) is a 584 amino acid protein with a molecular weight of approximately 62-72 kDa that functions as a transcriptional corepressor of IRF2 . This protein plays significant roles in immune response regulation, cell cycle progression, and has been implicated in various disease states including certain cancers. The biotin-conjugated antibody against IRF2BP1 provides researchers with a valuable tool for investigating these functions through various detection methods while leveraging the high-affinity avidin-biotin interaction system .

What applications are suitable for biotin-conjugated IRF2BP1 antibody?

The biotin-conjugated IRF2BP1 antibody is particularly useful for multiple laboratory techniques:

• ELISA (Enzyme-Linked Immunosorbent Assay): Primary application for quantitative detection of IRF2BP1

• Immunohistochemistry (IHC): Detection of IRF2BP1 in tissue sections with amplified signal through avidin-biotin complexes

• Immunofluorescence (IF): Cellular localization studies utilizing fluorophore-conjugated avidin or streptavidin as secondary detection reagents

• Western Blotting: When paired with appropriate avidin/streptavidin detection systems

• Immunoprecipitation: Utilizing the strong biotin-avidin interaction (Kd = 10^-15M) for protein pull-down experiments

The versatility stems from the unique properties of the avidin-biotin system, which allows for signal amplification and exceptional specificity .

How does biotinylation affect antibody performance compared to unconjugated versions?

Biotinylation offers several advantages but may introduce performance considerations:

Advantages:

• Signal amplification through avidin-biotin complexes, enhancing detection sensitivity

• Versatility across multiple detection platforms without requiring different secondary antibodies

• Stable complex formation unaffected by extremes of pH, temperature, and organic solvents

Potential Considerations:

• Over-biotinylation may affect antigen binding if modification occurs near the antigen-recognition site

• Steric hindrance from avidin-biotin complexes in densely packed epitopes

• Need for blocking endogenous biotin in certain tissues/cells

Researchers should validate biotinylated antibodies against unconjugated versions when transitioning methods. The biotin-conjugated IRF2BP1 antibody targeting amino acids 340-499 provides specific recognition of the human protein while offering the advantages of the biotin-avidin detection system .

What detection systems work best with biotin-conjugated IRF2BP1 antibody?

The optimal detection system depends on the application:

• For ELISA or Western blotting: Streptavidin-HRP or NeutrAvidin-HRP conjugates offer high sensitivity with low background. NeutrAvidin is particularly advantageous due to its deglycosylated nature, reducing nonspecific binding compared to native avidin .

• For Immunofluorescence: Fluorophore-conjugated streptavidin (Alexa Fluor dyes, FITC, etc.) provides excellent signal-to-noise ratios. NeutrAvidin fluorescent conjugates may provide enhanced performance due to lower nonspecific binding .

• For IHC applications: The streptavidin-biotin complex method offers signal amplification. For highest sensitivity, researchers can employ a high sensitivity NeutrAvidin-HRP conjugate which eliminates the lectin binding and high pI issues associated with native avidin .

The choice between avidin, streptavidin, or NeutrAvidin as the biotin-binding protein depends on the specific requirements of the experiment:

• NeutrAvidin (deglycosylated avidin) offers reduced nonspecific binding and lacks the RYD sequence that can cause nonspecific binding in IHC assays

• Streptavidin has a near-neutral pI but contains the RYG sequence that may create specificity issues in certain applications

• Native avidin is cost-effective but has higher nonspecific binding due to its high pI and lectin binding properties

What controls should be included when working with biotin-conjugated IRF2BP1 antibody?

A comprehensive experimental design should include:

• Positive Control: Known IRF2BP1-expressing samples (e.g., HeLa cells, HEK-293 cells, or Jurkat cells)

• Negative Controls:

  • Isotype control: Biotin-conjugated rabbit IgG matching the host species and isotype

  • Absence of primary antibody to assess background from the detection system

  • IRF2BP1-deficient or knockdown samples where possible

• Biotin-Specific Controls:

  • Endogenous biotin blocking: Pre-treatment with avidin or streptavidin followed by biotin to block endogenous biotin

  • Competition control: Pre-incubation with free biotin to validate specificity of detection system

• Cross-Reactivity Assessment:

  • Testing on samples from species not listed in the reactivity profile to validate specificity

  • When using mouse samples, ensure the detection system doesn't recognize endogenous mouse immunoglobulins

These controls help distinguish between specific IRF2BP1 detection and artifacts from the biotin-conjugation or detection system .

How can signal amplification be optimized using biotin-conjugated IRF2BP1 antibody?

Signal amplification using biotin-conjugated IRF2BP1 antibody can be optimized through several strategic approaches:

• Layered Avidin-Biotin Complex (ABC) Method:

  • Initial layer: Biotin-conjugated IRF2BP1 antibody binds to target

  • Secondary layer: Avidin/streptavidin with multiple biotin binding sites

  • Tertiary layer: Biotinylated reporter enzymes or fluorophores

  • This creates large complexes with multiple reporter molecules per epitope

• Tyramide Signal Amplification (TSA):

  • Combine biotin-conjugated antibody with avidin-HRP

  • Add biotinylated tyramide which becomes activated by HRP

  • Activated tyramide deposits biotin-tyramide conjugates near the reaction site

  • Apply additional avidin-conjugated reporters to the newly deposited biotin

• Optimizing Avidin/Streptavidin Selection:

  • For applications requiring minimal background: Use NeutrAvidin which lacks glycosylation and has lower nonspecific binding

  • For maximum signal strength: Consider using native avidin which has higher solubility but monitor background carefully

Verification of amplification without increasing background requires careful titration of all reagents and inclusion of appropriate controls to monitor signal-to-noise ratios.

What are the common pitfalls when using biotin-conjugated antibodies in multi-color immunofluorescence?

Multi-color immunofluorescence with biotin-conjugated IRF2BP1 antibody presents several challenges:

• Sequential Staining Requirements:

  • Complete the biotin-streptavidin detection before introducing additional biotinylated antibodies

  • Block all unoccupied biotin-binding sites with free biotin before introducing another biotinylated antibody

  • Consider using different conjugation systems (e.g., digoxigenin) for other markers

• Spectral Overlap Considerations:

  • Choose fluorophore-conjugated streptavidins with minimal spectral overlap with other fluorophores

  • Implement appropriate compensation controls when using flow cytometry

  • Consider linear unmixing algorithms for confocal microscopy

• Cross-Reactivity Issues:

  • Endogenous biotin in mitochondria and other organelles may cause background

  • Pre-block with avidin followed by biotin to neutralize endogenous biotin

  • Test tissues individually for endogenous biotin levels before designing complex panels

• Signal Balancing:

  • The strong amplification from avidin-biotin systems may overwhelm weaker signals

  • Titrate the biotin-conjugated IRF2BP1 antibody to match signal intensity with other markers

  • Consider using the biotin-conjugated antibody for the weakest expression target

Careful experimental design and sequential staining protocols can overcome these challenges to achieve reliable multi-color detection .

How can biotin-conjugated IRF2BP1 antibody be used for chromatin immunoprecipitation studies?

Chromatin immunoprecipitation (ChIP) using biotin-conjugated IRF2BP1 antibody offers advantages for studying IRF2BP1 protein-DNA interactions:

• Optimized Protocol Design:

  • Cross-link proteins to DNA using formaldehyde (typically 1% for 10 minutes)

  • Sonicate chromatin to 200-500bp fragments

  • Incubate sheared chromatin with biotin-conjugated IRF2BP1 antibody (ABIN7156619 or similar)

  • Capture complexes using streptavidin-coated magnetic beads rather than Protein A/G

  • Elute DNA and reverse cross-links for downstream analysis

• Advantages Over Conventional ChIP:

  • Higher sensitivity due to the strong avidin-biotin interaction (Kd = 10^-15M)

  • Reduced background from non-specific protein interactions

  • More consistent pull-down efficiency compared to Protein A/G-based systems

  • Compatible with stringent wash conditions without losing antibody-antigen binding

• Critical Optimization Parameters:

  • Sonication conditions must be optimized for target cell type

  • Biotin blocking in nuclear extracts may be necessary

  • Streptavidin bead capacity must be matched to expected quantity of biotinylated antibody

  • Consider pre-clearing lysates with streptavidin beads to reduce background

Published studies have successfully employed IRF2BP1 antibodies in ChIP applications, indicating the feasibility of this approach for investigating IRF2BP1's role in transcriptional regulation .

How do I validate species cross-reactivity for biotin-conjugated IRF2BP1 antibody?

The biotin-conjugated IRF2BP1 antibody targeting amino acids 340-499 (ABIN7156619) is reported to react with human samples . To validate reactivity or extend use to other species:

• Sequence Homology Analysis:

  • Compare the immunogen sequence (amino acids 340-499 of human IRF2BP1) with corresponding regions in target species

  • High homology (>85%) suggests potential cross-reactivity

  • Check for post-translational modifications that might differ between species

• Experimental Validation Methods:

  • Western blot using positive control lysates from multiple species

  • Include appropriate positive controls (human samples) and negative controls

  • Verify band appears at expected molecular weight (62-72 kDa for IRF2BP1)

  • Perform peptide competition assays to confirm specificity

• Progressive Testing Approach:

  • Begin with Western blot validation before attempting more complex applications

  • Use decreasing dilutions of antibody when testing new species

  • Compare staining patterns with published literature or alternative antibodies

For reference, other non-biotinylated IRF2BP1 antibodies show documented reactivity with human, mouse, and rat samples, suggesting conservation of epitopes across these species .

What are the expected expression patterns of IRF2BP1 across different tissues?

Understanding normal expression patterns is crucial for experimental design and interpretation:

• Tissue Distribution:

  • High expression: Lymphoid tissues, brain tissue, and reproductive organs

  • Positive Western blot detection has been reported in mouse brain tissue and rat lymph tissue

  • Human cell lines with confirmed expression include HeLa, HEK-293, and Jurkat cells

• Subcellular Localization:

  • Predominantly nuclear localization as expected for a transcriptional co-regulator

  • Can be visualized by immunofluorescence in HeLa cells

  • May show dynamic localization depending on cellular activation state

• Expression in Pathological Samples:

  • Detected in human breast cancer tissue by immunohistochemistry

  • Expression may be altered in inflammatory conditions due to its role in interferon signaling

When using the biotin-conjugated antibody, researchers should include positive control tissues/cells with known IRF2BP1 expression and compare staining patterns with unconjugated antibody versions to ensure biotinylation hasn't affected recognition properties.

How should sample preparation be modified for optimal IRF2BP1 detection using biotin-conjugated antibody?

Sample preparation significantly impacts detection quality with biotin-conjugated IRF2BP1 antibody:

• Protein Extraction Considerations:

  • For nuclear proteins like IRF2BP1, use nuclear extraction protocols

  • Include protease inhibitors to prevent degradation

  • For Western blotting, SDS-PAGE sample buffer should contain reducing agents

  • Avoid using milk as a blocking agent as it contains biotin that may interfere with detection

• Fixation for Microscopy:

  • For IHC: Suggested antigen retrieval with TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0)

  • For IF/ICC: 4% paraformaldehyde fixation followed by permeabilization with 0.1-0.5% Triton X-100

  • Methanol fixation may be preferable for nuclear antigen preservation

• Biotin-Specific Considerations:

  • Pre-block endogenous biotin with avidin/biotin blocking kit

  • For tissues high in endogenous biotin (kidney, liver, brain), consider alternative conjugation systems

  • When using biotin-rich culture media supplements, wash cells thoroughly before fixation

• Preparation for ELISA:

  • For sandwich ELISA, ensure capture antibody recognizes a different epitope than the biotin-conjugated antibody

  • Include biotin-free BSA in blocking buffers

  • Consider using streptavidin-coated plates to directly capture biotinylated samples

These modifications enhance signal specificity and reduce background interference from endogenous biotin or non-specific binding .

What advanced troubleshooting approaches can address weak or non-specific signals?

When encountering signal problems with biotin-conjugated IRF2BP1 antibody:

• Weak Signal Troubleshooting:

  • Increase antibody concentration (test range from 1:50 to 1:500 for IHC/IF)

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

  • Switch detection systems (try NeutrAvidin-HRP for higher sensitivity with lower background)

  • Enhance antigen retrieval (increase time or try alternative buffer systems)

  • Implement signal amplification (Tyramide Signal Amplification or multi-layer avidin-biotin complex)

• Non-specific Signal Reduction:

  • Use NeutrAvidin instead of avidin to eliminate lectin-mediated binding

  • Increase blocking stringency (5% BSA with 0.1-0.3% Triton X-100)

  • Pre-absorb antibody with tissue/cell homogenate from negative control species

  • Implement more stringent washing (increase wash times and detergent concentration)

  • Include additional blocking steps for endogenous biotin, peroxidases, and phosphatases

• Analytical Approaches:

  • Perform peptide competition assays to distinguish specific from non-specific binding

  • Compare staining patterns with alternative IRF2BP1 antibodies

  • Use IRF2BP1 knockdown/knockout samples as definitive negative controls

  • Employ panel of antibodies to multiple epitopes to confirm expression patterns

• Western Blot-Specific Optimization:

  • For heterogeneous band patterns (62-72 kDa range observed for IRF2BP1), investigate potential post-translational modifications or isoforms

  • Include phosphatase treatment to determine if band shifts are phosphorylation-dependent

  • Test gradient gels to improve separation of closely migrating isoforms

These approaches systematically address both sensitivity and specificity challenges while maintaining the advantages of the biotin-avidin detection system .

How can biotin-conjugated IRF2BP1 antibody be utilized in multiplexed protein detection systems?

Biotinylated IRF2BP1 antibody offers versatile options for multiplexed detection:

• Sequential Multiplexing Strategies:

  • Complete IRF2BP1 detection with streptavidin-conjugate of choice

  • Block remaining biotin-binding sites with excess free biotin

  • Proceed with non-biotin detection systems (direct fluorophore conjugates or alternative hapten systems)

  • Can be combined with spectral imaging for increased multiplexing capacity

• Mass Cytometry (CyTOF) Applications:

  • Conjugate streptavidin with rare earth metals

  • Detect biotinylated IRF2BP1 antibody in combination with >30 other markers

  • Especially valuable for investigating IRF2BP1's role in complex immune cell populations

• Proximity Ligation Assay (PLA):

  • Combine biotin-conjugated IRF2BP1 antibody with proximity probes

  • Investigate protein-protein interactions between IRF2BP1 and potential binding partners

  • Provides spatial resolution of molecular interactions within cells

• Imaging Mass Cytometry:

  • Use metal-tagged streptavidin for detection of biotinylated IRF2BP1 antibody

  • Combine with multiple other antibodies for highly multiplexed tissue imaging

  • Allows correlation of IRF2BP1 expression with tissue architecture and cellular context

These approaches leverage the biotin-conjugation to integrate IRF2BP1 detection into complex experimental designs while maintaining specificity and sensitivity .

What experimental designs can investigate the role of IRF2BP1 in transcriptional regulation?

To study IRF2BP1's function as a transcriptional corepressor:

• ChIP-Seq Approach:

  • Perform ChIP with biotin-conjugated IRF2BP1 antibody and streptavidin beads

  • Sequence immunoprecipitated DNA to identify genome-wide binding sites

  • Integrate with RNA-Seq and epigenetic data to correlate binding with gene regulation

  • Compare binding patterns under different cellular conditions or stimuli

• Protein Complex Analysis:

  • Use biotinylated IRF2BP1 antibody for immunoprecipitation

  • Identify co-precipitating proteins by mass spectrometry

  • Validate interactions using reciprocal IPs and proximity ligation assays

  • Map domains involved in protein-protein interactions

• Functional Genomics Integration:

  • Combine IRF2BP1 ChIP data with CRISPR screens

  • Identify genes functionally regulated by IRF2BP1 binding

  • Correlate binding patterns with phenotypic outcomes

  • Create network models of IRF2BP1-mediated transcriptional regulation

• Dynamic Regulation Studies:

  • Track IRF2BP1 binding in response to interferon stimulation

  • Use time-course experiments to map temporal dynamics of complex assembly

  • Correlate with post-translational modifications and protein turnover

  • Implement live-cell imaging techniques with complementary fluorescent tags

These experimental approaches leverage the specificity of the biotin-conjugated antibody while integrating multiple techniques to comprehensively investigate IRF2BP1's regulatory functions.

How can spatial protein profiling be achieved using biotin-conjugated IRF2BP1 antibody?

Spatial protein profiling with biotin-conjugated IRF2BP1 antibody enables examination of expression patterns in cellular context:

• Advanced Microscopy Approaches:

  • Super-resolution microscopy: Use small fluorophore-conjugated streptavidin (Alexa Fluor dyes) for nanoscale localization

  • Expansion microscopy: Physically expand samples after IRF2BP1 labeling to achieve sub-diffraction resolution

  • CODEX multiplexed imaging: Incorporate biotinylated IRF2BP1 antibody into cyclic immunofluorescence panels

• Spatial Transcriptomics Integration:

  • Combine IRF2BP1 protein detection with in situ transcriptomics

  • Correlate protein localization with local transcriptional landscapes

  • Investigate spatial relationships between IRF2BP1 and its target genes

• Tissue Microenvironment Analysis:

  • Map IRF2BP1 expression across different cell types within complex tissues

  • Correlate with cell state markers and tissue architecture

  • Implement neighborhood analysis to identify spatial associations

• Intracellular Compartment Profiling:

  • Co-stain with markers for nuclear substructures (nucleoli, splicing speckles, etc.)

  • Investigate potential shuttling between nuclear and cytoplasmic compartments

  • Study redistribution upon cellular activation or stress

The biotin-conjugated antibody provides flexibility across these platforms while maintaining consistent epitope recognition, allowing for comparative studies across different spatial scales and experimental systems .