BIR2 Antibody

What is BIR2/BIRC2 and what cellular functions does it regulate?

BIR2/BIRC2 (Baculoviral IAP Repeat Containing 2) is a member of the inhibitor of apoptosis protein (IAP) family that regulates fundamental cell death and survival signaling pathways . It contains multiple BIR (Baculoviral IAP Repeat) domains that mediate protein-protein interactions. BIRC2 plays critical roles in:

• Regulation of cell death and apoptosis

• Immune signaling pathways, particularly interferon and defense response signaling

• Cell-cycle regulation and DNA damage repair processes

• Nuclear functions through interaction with histone H3

Research shows that BIRC2 depletion significantly affects genes involved in interferon response, defense signaling pathways, and cell-cycle regulation as determined by RNA-seq analysis .

What detection methods are most effective when working with BIR2 antibodies?

When working with BIR2 antibodies, several detection methods have proven effective:

• ELISA (Enzyme-Linked Immunosorbent Assay): Sandwich ELISA methods can reliably detect BIRC2 with sensitivity in the pg/mL range (78.125 pg/mL minimum detection limit for some commercial kits)

• Western Blotting: Effective for analyzing protein expression levels and post-translational modifications

• Immunoprecipitation: Useful for studying protein-protein interactions, particularly between BIR2 and binding partners like RIPK2

• Immunohistochemistry: For tissue localization studies

• Fluorescence microscopy: For cellular localization studies

The choice of detection method should align with your specific research question. For quantitative detection of BIRC2 in serum, plasma, or tissue homogenates, colorimetric sandwich ELISA provides high sensitivity and excellent specificity .

What sample types can be effectively analyzed using BIR2 antibodies?

BIR2 antibodies have been successfully used to analyze multiple sample types:

• Serum and plasma samples: Effective for clinical research and biomarker studies

• Tissue homogenates: Useful for examining expression in different organs and tissues

• Cell lysates: For in vitro studies of protein expression and regulation

• Primary cells: Including peripheral blood mononuclear cells (PBMCs) from patients with XIAP mutations

• Reconstituted cell lines: Such as XIAP-deficient cell lines used to study BIR2 domain function

Different sample preparation protocols may be required depending on the sample type. For instance, tissue homogenates typically require mechanical disruption and protein extraction buffers, while serum samples may require dilution to minimize matrix effects in assays.

How can BIR2 antibodies be used to study XIAP mutations in XLP2?

BIR2 antibodies are valuable tools for studying X-linked lymphoproliferative syndrome type-2 (XLP2) caused by mutations in the XIAP BIR2 domain. Methodological approaches include:

• Analyzing protein-protein interactions: BIR2 antibodies can help assess how XLP2-associated mutations affect interactions between XIAP and RIPK2. Co-immunoprecipitation experiments have shown that XLP2-BIR2 mutations (including R166I, L207P, and V198M) abolish the XIAP-RIPK2 interaction .

• Evaluating ubiquitylation activity: XLP2-BIR2 mutations impair XIAP-mediated ubiquitylation of RIPK2. Using BIR2 antibodies in ubiquitylation assays allows researchers to track how mutations affect this process .

• Assessing NOD2 signaling: BIR2 antibodies can be used to investigate how XLP2-BIR2 mutations impair NOD1/2-dependent immune signaling in:

  • Reconstituted XIAP-deficient cell lines

  • Primary cells from XLP2 patients

• Patient sample analysis: BIR2 antibodies enable comparative studies between healthy donors and XLP2 patients to identify functional defects in NOD2-dependent immune responses .

What approaches are recommended when studying BIR2-RIPK2 interactions?

When investigating BIR2-RIPK2 interactions, consider these methodological approaches:

• Co-immunoprecipitation studies: Use BIR2 antibodies to pull down protein complexes and analyze RIPK2 association. Research has shown that the XIAP BIR2 domain is crucial for RIPK2 binding, and XLP2-BIR2 mutations abolish this interaction .

• Domain mapping experiments: Utilize truncated constructs to identify specific interaction regions. Studies confirm that the interaction between XIAP and RIPK2 requires the XIAP BIR2 domain and does not involve other domains of XIAP .

• Mutational analysis: Introduce specific mutations in the BIR2 domain to identify critical residues for RIPK2 binding. Six XLP2-BIR2 mutations have been shown to abrogate co-purification of endogenous RIPK2 with XIAP .

• Functional assays: Measure downstream signaling (e.g., NF-κB activation) to assess the functional consequences of disrupted BIR2-RIPK2 interactions. XIAP XLP2-BIR2 variants are unable to restore NOD2-induced NF-κB activation in XIAP-deficient cells .

• Inhibitor studies: Use Smac mimetic compounds (SMCs) to competitively disrupt BIR2-RIPK2 interactions and analyze the functional outcomes .

How can I use BIR2 antibodies to investigate cancer immunology?

BIR2 antibodies are valuable tools in cancer immunology research, particularly given BIRC2's role in immune evasion mechanisms. Here's how to approach this research:

• Analysis of tumor immune microenvironment:

  • Use BIR2 antibodies to assess BIRC2 expression in tumor cells and correlate with immune cell infiltration patterns

  • Research has shown that BIRC2 expression impairs anti-cancer immunity and immunotherapy response

• Knockdown/knockout validation studies:

  • Use BIR2 antibodies to confirm successful BIRC2 depletion in knockdown/knockout experiments

  • Studies demonstrate that knockdown of BIRC2 in mouse melanoma or breast cancer cells increases expression of chemokine CXCL9 and impairs tumor growth by increasing intratumoral activated CD8+ T cells

• Combination therapy assessment:

  • Monitor BIRC2 expression during immune checkpoint blockade (ICB) treatment

  • Investigate whether combining BIRC2 inhibition with ICB enhances therapeutic response

• Immune signaling pathway analysis:

  • Use BIR2 antibodies in phospho-flow cytometry or western blotting to assess activation of immune signaling pathways

  • RNA-seq analysis reveals that genes involved in interferon and defense response signaling are significantly affected by BIRC2 depletion

How can I analyze BIR2's role in DNA damage repair pathways?

To investigate BIR2's involvement in DNA damage repair pathways, consider these methodological approaches:

• DNA damage induction and repair kinetics:

  • Use BIR2 antibodies to track BIRC2 expression and localization during DNA damage response

  • Induce DNA damage with agents like etoposide and monitor repair over time

  • Research shows that overexpression of BIRC2 delays DNA damage repair, as evidenced by persistent levels of γH2AX even 6 hours after drug removal

• Cell cycle analysis in relation to BIRC2 expression:

  • Use flow cytometry with BIR2 antibodies to correlate BIRC2 expression with cell cycle phases

  • Overexpression of BIRC2 has been shown to delay recovery of normal cell cycle progression after DNA damage

• Chromatin immunoprecipitation (ChIP):

  • Use BIR2 antibodies for ChIP assays to identify BIRC2 binding sites on chromatin during DNA damage response

  • BIRC2 BIR2 and BIR3 domains have been shown to interact with histone H3 tail

• Co-immunoprecipitation with DNA repair proteins:

  • Identify BIRC2 interactions with DNA repair machinery components

  • Map these interactions to specific phases of the DNA damage response

What are the optimal protocols for using BIR2 antibodies in ELISA?

When using BIR2 antibodies in ELISA, follow these optimized protocols for reliable results:

• Sample preparation:

  • For serum/plasma: Dilute samples appropriately in the provided sample dilution buffer

  • For tissue homogenates: Process tissues in homogenization buffer, centrifuge to remove debris, and quantify protein concentration

  • Perform all sample preparation steps on ice to prevent protein degradation

• Procedure for sandwich ELISA (based on commercial kit protocols):

  • Add 100 μL standard or prepared sample to each well of the pre-coated plate

  • Incubate for 90 minutes at 37°C

  • Aspirate and wash plates 2 times with wash buffer

  • Add 100 μL Biotin-labeled antibody working solution

  • Continue with detection steps as per kit instructions

• Quality control measures:

  • Include blank, negative controls, and positive controls in each assay

  • Generate a standard curve using purified BIRC2 protein (standard concentration range: 78.125 pg/mL - 5000 pg/mL)

  • Perform all samples in duplicate or triplicate for statistical validity

• Assay performance characteristics:

  • Sensitivity: Typically around 46.875 pg/mL for high-quality ELISA kits

  • Detection range: 78.125 pg/mL - 5000 pg/mL

  • Minimal cross-reactivity with analogous proteins should be expected

How should I optimize immunoprecipitation experiments with BIR2 antibodies?

For successful immunoprecipitation experiments with BIR2 antibodies, follow these optimization strategies:

• Cell lysis conditions:

  • Use gentle lysis buffers that preserve protein-protein interactions (e.g., RIPA buffer with protease inhibitors)

  • For nuclear interactions (such as BIRC2-histone H3), include specific nuclear extraction protocols

  • Optimize detergent concentration to maintain protein interactions while solubilizing membrane proteins

• Antibody selection and validation:

  • Validate antibody specificity using western blotting before immunoprecipitation

  • For studying specific domains (like BIR2 or BIR3), use domain-specific antibodies

  • Consider using epitope-tagged constructs when studying mutated versions of BIRC2/XIAP

• Control experiments:

  • Include IgG control to identify non-specific binding

  • Use BIRC2/XIAP knockout or knockdown samples as negative controls

  • For mutation studies, compare wild-type and mutant variants (e.g., XLP2-BIR2 mutations)

• Detection of interacting partners:

  • For known interactions (like BIRC2-RIPK2), use specific antibodies against the partner protein

  • For novel interactions, consider mass spectrometry analysis of immunoprecipitates

  • When studying ubiquitylation events, include deubiquitinase inhibitors in lysis buffers

Research has shown that all six XLP2-BIR2 mutations abrogate the co-purification of endogenous RIPK2 with XIAP, while binding to other partners like TAB1 remains unaffected .

How can I use BIR2 antibodies to assess the effects of small molecule inhibitors?

BIR2 antibodies are essential tools for evaluating how small molecule inhibitors affect BIRC2 function:

• Monitoring target engagement:

  • Use pull-down assays with BIR2 antibodies to determine if inhibitors disrupt specific protein-protein interactions

  • NMR studies show that the small molecule inhibitor LCL161 targets BIRC2 BIR2 and BIR3 domains but not BIR1, with binding affinities of 2.2 μM for BIR2 and 490 nM for BIR3

• Functional outcome assessment:

  • Use BIR2 antibodies in western blotting to examine how inhibitors affect downstream signaling pathways

  • For nuclear interactions, investigate whether inhibitors like LCL161 disrupt BIRC2 association with histone H3

• Cellular localization studies:

  • Employ immunofluorescence with BIR2 antibodies to track changes in BIRC2 localization after inhibitor treatment

  • LCL161 has been shown to disrupt the association of endogenous BIRC2 with H3

• Protein degradation analysis:

  • Monitor BIRC2 levels after inhibitor treatment to determine if targeted degradation occurs

  • LCL161 mediates degradation of BIRC2 in HIV-1-infected human CD4+ T cells

• Combination therapy studies:

  • Use BIR2 antibodies to assess BIRC2 status when combining small molecule inhibitors with other therapeutic approaches

  • LCL161 shows potent anticancer activity in numerous tumor types and can sensitize cancer cells to conventional chemotherapy

What controls should I include when using BIR2 antibodies in cancer research?

When conducting cancer research with BIR2 antibodies, include these essential controls:

• Expression level controls:

  • Cell lines with known BIRC2 expression levels (high, medium, low)

  • BIRC2 knockout or knockdown cells created using CRISPR-Cas9 or siRNA

  • Matched normal and tumor tissues to establish baseline expression differences

• Functional validation controls:

  • For immune response studies, include positive controls like immune stimulants (e.g., IFNγ)

  • In macrophage studies, compare BIRC2 inhibition effects on cytokine production (e.g., IL-6 production)

  • For apoptosis studies, include known apoptosis inducers as positive controls

• Antibody validation controls:

  • Peptide blocking experiments to confirm antibody specificity

  • Secondary antibody-only controls to identify non-specific binding

  • Multiple antibodies targeting different epitopes of BIRC2 to confirm results

• Treatment response controls:

  • For inhibitor studies, include dose-response curves

  • Time-course experiments to determine optimal treatment durations

  • Combined marker analysis (e.g., BIRC2 and CXCL9 expression in cancer cells)

• Patient sample controls:

  • Age and gender-matched normal samples

  • Samples spanning different cancer stages and grades

  • Treatment-naive versus post-treatment samples

Research has shown that knockdown of BIRC2 in mouse melanoma or breast cancer cells increases intratumoral activated CD8+ T cells, providing a methodological framework for studying BIRC2's role in cancer immune evasion .

How can I address non-specific binding when using BIR2 antibodies?

Non-specific binding is a common challenge when working with antibodies. Here are methodological solutions specifically for BIR2 antibody research:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Consider commercial blocking buffers formulated to reduce background

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • For ELISA applications, follow the manufacturer's recommended dilution for biotin-labeled antibodies

  • For western blotting, typically start with 1:1000 dilution and adjust as needed

• Sample preparation refinement:

  • For tissue homogenates, ensure complete lysis and removal of debris

  • For serum/plasma, consider pre-clearing with protein A/G beads

  • Use appropriate sample dilution buffer to minimize matrix effects

• Washing protocol optimization:

  • Increase number and duration of wash steps

  • Ensure complete removal of wash buffer between steps

  • Use appropriate wash buffer with optimal detergent concentration

• Cross-reactivity assessment:

  • Test antibody against recombinant BIRC family members to assess specificity

  • Note that some cross-reactivity with BIRC2 analogues may still exist despite optimization

What are the key considerations when designing experiments to study BIR2 domain mutations?

When investigating BIR2 domain mutations, particularly in the context of XLP2, consider these methodological approaches:

• Mutation selection and validation:

  • Include clinically relevant mutations (e.g., R166I, L207P, V198M) identified in XLP2 patients

  • Verify mutant protein expression levels before functional studies

  • Create stable cell lines expressing wild-type and mutant variants for consistent comparisons

• Functional readouts:

  • NF-κB reporter assays to measure NOD1/2-dependent signaling

  • NOD2 ligand stimulation (e.g., L18-MDP) to assess pathway activation

  • Monitor ubiquitylation of RIPK2 as a direct readout of XIAP BIR2 function

• Interaction studies:

  • Design co-immunoprecipitation experiments to assess how mutations affect binding to known partners like RIPK2

  • Include TAB1 interaction analysis as a control (binds to BIR1 domain, should be unaffected by BIR2 mutations)

  • Consider proximity ligation assays for in situ detection of protein interactions

• Comparative analysis framework:

  • Always compare mutant variants to wild-type protein in the same experimental setting

  • Include the F495A mutation as a control for XIAP's E3 ligase activity

  • For patient-derived cells, compare to age-matched healthy donor samples

Research has demonstrated that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signaling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines .

How do I interpret contradictory results between different BIR2 antibody detection methods?

When faced with contradictory results across different detection methods, use this systematic approach:

• Method-specific factors assessment:

  • ELISA: Consider detection limits (typically 78.125 pg/mL for BIRC2), sample matrix effects, and potential cross-reactivity

  • Western blotting: Evaluate protein denaturation effects, epitope accessibility, and transfer efficiency

  • Immunoprecipitation: Assess buffer conditions that might disrupt protein-protein interactions

• Antibody characteristics comparison:

  • Epitope location: Different antibodies may target distinct regions of BIRC2

  • Clonality: Monoclonal vs. polyclonal antibodies may yield different results

  • Affinity and specificity: Higher affinity antibodies may detect lower expression levels

• Biological context analysis:

  • Cell/tissue type differences: BIRC2 expression and function varies across tissues

  • Experimental conditions: Stress, cytokine stimulation, or drug treatments may affect BIRC2 detection

  • Post-translational modifications: May alter epitope recognition

• Validation strategies:

  • Use multiple antibodies targeting different epitopes

  • Employ genetic approaches (siRNA, CRISPR) to confirm specificity

  • Include recombinant BIRC2 protein as a positive control

• Data integration approach:

  • Consider each method's strengths and limitations

  • Weigh results based on technical quality and reproducibility

  • Develop a consensus interpretation that accounts for methodological differences

How can BIR2 antibodies be used to study the role of BIRC2 in cancer immunotherapy resistance?

BIR2 antibodies offer powerful tools for investigating BIRC2's role in cancer immunotherapy resistance:

• Tumor microenvironment profiling:

  • Use BIR2 antibodies in multiplex immunohistochemistry to correlate BIRC2 expression with immune cell infiltration patterns

  • Research has shown that BIRC2 expression impairs anti-cancer immunity and promotes resistance to immune checkpoint blockade (ICB)

• Mechanistic studies:

  • Investigate how BIRC2 affects chemokine expression (particularly CXCL9) using knockdown experiments validated with BIR2 antibodies

  • Analyze how BIRC2 modulates CD8+ T cell infiltration and activation in tumors

• Biomarker development:

  • Use BIR2 antibodies to assess BIRC2 expression in patient tumor samples before and during immunotherapy

  • Correlate expression levels with treatment response to determine predictive value

• Combination therapy design:

  • Monitor BIRC2 expression during treatment with BIRC2 inhibitors combined with immune checkpoint inhibitors

  • Use BIR2 antibodies to confirm target engagement in preclinical studies

• Resistance mechanism identification:

  • Compare BIRC2 expression and localization in immunotherapy-responsive versus resistant tumors

  • Investigate downstream signaling pathways affected by BIRC2 that contribute to resistance

Research demonstrating that knockdown of BIRC2 expression in mouse melanoma or breast cancer cells impairs tumor growth by increasing intratumoral activated CD8+ T cells provides a foundation for these studies .

What are the newest methodologies for studying BIRC2 BIR3-histone H3 interactions?

Recent advances have enabled more detailed study of BIRC2 BIR3-histone H3 interactions:

• Structural analysis techniques:

  • X-ray crystallography and cryo-EM to determine the precise structure of the BIRC2 BIR3-H3 complex

  • NMR spectroscopy to study binding dynamics and identify critical interaction residues

  • Studies have reported the structure of the BIRC2 BIR3-H3 complex, providing insights into molecular interactions

• ChIP-sequencing applications:

  • Use BIR2 antibodies in ChIP-seq to map genome-wide binding sites of BIRC2

  • Integrate with histone modification maps to understand the functional context of binding

  • Correlate with gene expression data to identify regulated genes

• CRISPR-based screening:

  • Deploy CRISPR screens to identify genes that modulate BIRC2-H3 interactions

  • Use BIR2 antibodies to validate hit genes that affect nuclear localization or function

• Live-cell imaging approaches:

  • Utilize fluorescently tagged BIRC2 constructs combined with H3 markers to visualize interactions in real-time

  • Apply FRET or BRET techniques to measure protein-protein proximity in living cells

• Small molecule intervention:

  • Use BIR2 antibodies to assess how inhibitors like LCL161 disrupt BIRC2-H3 interactions

  • Research has shown that LCL161 disrupts the association of endogenous BIRC2 with H3 and stimulates cell death in cancer cells