BIRC8 Antibody

What is BIRC8 and what makes it distinct from other IAP family members?

BIRC8, also known as ILP-2, is a homologous protein to BIRC4 that functions as an inhibitor of apoptosis protein. Unlike other IAP family members that directly inhibit caspase activity, BIRC8 operates through a distinctive mechanism by impeding apoptosis induced by BAX without directly inhibiting the activity of caspases . The protein is encoded by a gene located on chromosome 19q13.42 and consists of a single exon .

BIRC8 is predicted to enable cysteine-type endopeptidase inhibitor activity involved in apoptotic processes and ubiquitin protein ligase activity. Additionally, it is involved in the negative regulation of apoptotic processes, positive regulation of canonical Wnt signaling pathway, and positive regulation of protein ubiquitination . Its activity has been predicted in both cytoplasmic and nuclear compartments, suggesting potential multifunctional roles in cellular homeostasis.

What are the standard applications for BIRC8 antibodies in research?

BIRC8 antibodies are versatile research tools validated for multiple applications in molecular and cellular biology. Based on available commercial antibodies, researchers commonly employ these antibodies in the following techniques:

When designing experiments, researchers should be aware that optimal dilutions may be sample-dependent and require titration for each specific testing system to obtain optimal results .

How is BIRC8 gene classified and what are its key identifiers in genomic databases?

BIRC8 is currently classified as a pseudogene in some databases, though it demonstrates functional properties in specific contexts. Researchers investigating BIRC8 at the genomic level should reference the following identifiers:

Researchers should note that information about BIRC8 continues to evolve, with recent updates to genomic databases as of March 2025 . When conducting literature searches or database queries, using all known synonyms increases the likelihood of comprehensive results.

What are the most effective methods for validating BIRC8 antibody specificity?

Validating antibody specificity is crucial for generating reliable research data. For BIRC8 antibodies, researchers should implement the following validation approaches:

• Peptide competition assays: Since BIRC8 antibodies are often raised against synthetic peptides corresponding to amino acids near the amino terminus of human ILP-2 , synthetic competing peptides can confirm binding specificity.

• Knockout/knockdown controls: Using CRISPR-Cas9 BIRC8 knockout cells or siRNA-mediated knockdown samples as negative controls when performing Western blot or immunofluorescence.

• Multiple antibody validation: Comparing results using antibodies targeting different epitopes of BIRC8. Commercial antibodies are available that target different regions, such as AA 1-236 and AA 141-236 .

• Cross-reactivity assessment: Testing the antibody against samples from multiple species (human, mouse, rat) to confirm the expected reactivity pattern based on the manufacturer's specifications .

• Molecular weight verification: Confirming that the detected protein band appears at the expected molecular weight of approximately 30-35 kDa, which corresponds to the calculated weight of BIRC8 isoforms .

What experimental considerations are important when investigating BIRC8 in cancer models?

When studying BIRC8 in cancer research, several methodological considerations should be addressed:

• Tissue selection: BIRC8 expression has been detected in various cancers, including breast carcinoma, hematological neoplasms, hepatocellular carcinoma, nasopharyngeal carcinoma, and neuroblastoma . Researchers should carefully select appropriate positive and negative control tissues.

• Expression heterogeneity: Expression patterns may vary significantly between cancer subtypes and even within the same tumor. Multiple sampling and quantitative approaches are recommended.

• Normal tissue comparison: Include normal counterpart tissues, particularly testis and lymphoblastic cells where BIRC8 has been detected normally , to establish differential expression patterns.

• Functional context: Design experiments that can distinguish between BIRC8's anti-apoptotic function and its potential roles in other cellular processes like Wnt signaling pathway regulation .

• Antibody selection: Choose antibodies with validated reactivity in the specific cancer model being studied. Some antibodies have documented reactivity with human and mouse samples , which is important for translational research using mouse xenograft models.

How should researchers optimize Western blot protocols for detecting BIRC8?

Optimizing Western blot protocols for BIRC8 detection requires attention to several specific parameters:

• Sample preparation: Use appropriate lysis buffers that effectively solubilize both cytoplasmic and nuclear proteins, as BIRC8 is predicted to be active in both compartments .

• Protein loading: Load 20-40 μg of total protein per lane, as BIRC8 may be expressed at relatively low levels in some tissues.

• Gel percentage: Use 10-12% polyacrylamide gels to achieve optimal separation of proteins in the 30-35 kDa range where BIRC8 is typically detected .

• Antibody dilution: Begin with the manufacturer's recommended dilution (typically 1:500-1:1000 for Western blot ) and adjust based on signal-to-noise ratio.

• Blocking conditions: Test both milk-based and BSA-based blocking buffers, as some BIRC8 epitopes may be obscured by certain blocking agents.

• Detection sensitivity: Consider using enhanced chemiluminescence (ECL) with longer exposure times if signal is weak, as BIRC8 expression levels may vary between tissue types.

• Positive controls: Include brain tissue samples from human or mouse, which have been documented to express detectable levels of BIRC8 .

How can researchers effectively distinguish between BIRC8 and other IAP family members in functional studies?

Distinguishing between BIRC8 and other IAP family members requires strategic experimental design:

• Functional readouts: Unlike other IAPs that directly inhibit caspases, BIRC8 primarily blocks BAX-induced apoptosis . Design assays that distinguish between these mechanisms, such as caspase activity assays paired with BAX translocation studies.

• Domain-specific interventions: Utilize constructs expressing specific domains of BIRC8 to identify which regions are responsible for its unique functions compared to other IAPs.

• Interaction studies: Implement co-immunoprecipitation experiments to identify BIRC8-specific protein interactions that differ from those of other IAP family members.

• Subcellular localization: Track the subcellular distribution of BIRC8 using immunofluorescence with confocal microscopy, as its localization patterns may differ from other IAPs.

• Knockout/knockdown specificity: When designing genetic perturbation experiments, verify that siRNAs or guide RNAs target BIRC8 specifically without affecting other IAP family members with sequence homology.

What are the current challenges in interpreting BIRC8 expression data across different tumor types?

Researchers face several challenges when interpreting BIRC8 expression data in cancer studies:

• Inconsistent detection methods: Different studies employ varied techniques (IHC, WB, qPCR) with different antibodies and probes, making cross-study comparisons difficult.

• Context-dependent functions: BIRC8's role may differ between cancer types due to interactions with tissue-specific factors. This contextual function requires careful experimental design that accounts for tissue-specific microenvironments.

• Correlation versus causation: While BIRC8 expression has been reported in multiple cancer types , establishing its causative role in tumorigenesis requires functional studies beyond expression correlation.

• Isoform-specific effects: Potential isoforms or post-translational modifications of BIRC8 may exhibit different functions across tumor types. Researchers should employ methods that can distinguish between these variants.

• Prognostic significance validation: The specific implications of BIRC8 expression for treatment outcomes and prognosis require further evaluation through properly powered studies with appropriate statistical methods .

How does BIRC8's interaction with the Wnt signaling pathway impact experimental design in cancer research?

BIRC8 has been predicted to positively regulate the canonical Wnt signaling pathway , which has significant implications for experimental design:

• Dual pathway analysis: Experiments should simultaneously assess both apoptotic regulation and Wnt signaling components to understand how BIRC8 may coordinately regulate these pathways.

• β-catenin interaction studies: Design co-immunoprecipitation or proximity ligation assays to determine whether BIRC8 directly interacts with β-catenin or other Wnt pathway components.

• Reporter assays: Implement TCF/LEF luciferase reporter assays to quantify the impact of BIRC8 expression or knockdown on Wnt pathway activation.

• Genetic interaction models: Use combinatorial knockdown/knockout approaches targeting both BIRC8 and key Wnt pathway components to identify synthetic interactions.

• Cancer model selection: Choose cancer models where Wnt signaling plays a well-established role (e.g., colorectal cancer) to effectively study the potential dual functionality of BIRC8.

What are common troubleshooting strategies when BIRC8 antibodies produce unexpected results?

When researchers encounter unexpected results with BIRC8 antibodies, the following troubleshooting strategies are recommended:

• Epitope accessibility issues: If signal is weak despite optimized protocols, consider alternative sample preparation methods that may better expose the epitope. BIRC8 antibodies are commonly raised against amino-terminal epitopes , which may be obscured in certain conformations.

• Non-specific binding: If multiple bands appear in Western blots, optimize blocking conditions and antibody concentration. Consider using antibodies raised against different epitopes for confirmation, such as those targeting amino acids 1-236 versus 141-236 .

• Species cross-reactivity discrepancies: If the antibody doesn't perform as expected across species, review the immunogen sequence. Some BIRC8 antibodies are raised against human-specific sequences that may have limited homology with other species.

• Negative results in expected positive samples: For tissues where BIRC8 is expected but not detected, consider enrichment techniques such as immunoprecipitation before detection or more sensitive detection systems.

• Signal variability between experiments: Implement rigorous standardization of protocols, including consistent sample handling, lysis conditions, and incubation times. Use internal loading controls appropriate for the subcellular fraction being analyzed.

What considerations are important when selecting between different fluorophore-conjugated BIRC8 antibodies for imaging studies?

Selection of appropriate fluorophore-conjugated BIRC8 antibodies requires consideration of multiple factors:

• Spectral compatibility: Choose fluorophores that are compatible with your imaging system and other fluorophores being used in multiplexed experiments. Options include DyLight 755 and various AbBy Fluor® conjugates (594, 680, 555) .

• Tissue autofluorescence: Consider tissue-specific autofluorescence profiles when selecting fluorophores. For highly autofluorescent tissues, choose fluorophores in the far-red spectrum such as DyLight 755.

• Subcellular localization: Since BIRC8 is predicted to be active in both cytoplasm and nucleus , select fluorophores with appropriate brightness and photostability for capturing potentially subtle differences in subcellular distribution.

• Application-specific needs: For live-cell imaging, consider pH-stable fluorophores, while for fixed tissues or cells, photostability may be the priority.

• Quantitative imaging requirements: For quantitative applications, select fluorophores with minimal photobleaching and consistent quantum yield. Calibrate the imaging system using appropriate standards for the selected fluorophore.

How should researchers interpret contradictory findings between different detection methods for BIRC8?

When confronted with contradictory findings between different BIRC8 detection methods, researchers should:

• Evaluate methodological differences: Different techniques (WB, ELISA, IF, IHC) have distinct sensitivity thresholds and may detect different epitopes or conformational states of BIRC8.

• Consider isoform detection: Determine whether the contradictory results might represent detection of different BIRC8 isoforms. BIRC8 has a predicted calculated molecular weight of 27 kDa (236aa) or 39 kDa (338aa), but is typically observed at 30-35 kDa .

• Validate with orthogonal approaches: Implement multiple, methodologically distinct techniques to triangulate findings. For example, combine protein detection (WB, IP) with transcript analysis (RT-qPCR, RNA-seq).

• Control for sample preparation artifacts: Different sample preparation methods may alter epitope accessibility or protein solubility. Standardize preparation methods or test multiple approaches.

• Consider biological variables: Cell type, differentiation state, and experimental conditions may influence BIRC8 expression and localization. Design experiments with appropriate biological controls to account for these variables.

What emerging technologies might enhance the study of BIRC8's role in regulating protein ubiquitination?

BIRC8 is predicted to play a role in positive regulation of protein ubiquitination , suggesting several promising research directions using emerging technologies:

• Proximity-dependent biotinylation (BioID or TurboID): These techniques can identify proteins that come into close proximity with BIRC8, potentially revealing substrates for its ubiquitin ligase activity.

• Ubiquitin proteomics: Quantitative ubiquitinomics using mass spectrometry to compare ubiquitination patterns in BIRC8-expressing versus BIRC8-knockout cells.

• PROTAC approaches: Proteolysis targeting chimeras could be designed to target BIRC8, allowing for rapid, inducible degradation to study acute effects on cellular ubiquitination landscapes.

• Live-cell ubiquitination sensors: Fluorescent biosensors for ubiquitination could be employed to visualize BIRC8-dependent ubiquitination events in real-time.

• CryoEM structural studies: Structural determination of BIRC8 in complex with its substrates and ubiquitination machinery could reveal mechanistic insights into its function.

How might single-cell techniques advance our understanding of BIRC8 expression heterogeneity in cancer?

Single-cell approaches offer unprecedented opportunities to understand BIRC8's role in cancer heterogeneity:

• Single-cell RNA sequencing: This technique can reveal cell-specific BIRC8 expression patterns within heterogeneous tumors, potentially identifying specific cell populations where BIRC8 plays critical roles.

• Single-cell proteomics: Mass cytometry (CyTOF) with BIRC8 antibodies can quantify protein expression at the single-cell level alongside other cancer-relevant markers.

• Spatial transcriptomics: These methods can map BIRC8 expression patterns within the tumor microenvironment, potentially revealing relationships with specific architectural features or infiltrating immune cells.

• Lineage tracing with BIRC8 reporters: Genetic lineage tracing systems driven by the BIRC8 promoter could track the fate of BIRC8-expressing cells during tumor evolution.

• Single-cell functional genomics: CRISPR perturbation of BIRC8 combined with single-cell readouts can identify cell-specific dependencies and functions that may be obscured in bulk analyses.

What are the most promising therapeutic approaches targeting BIRC8 in cancer based on current research?

Based on current understanding of BIRC8's functions, several therapeutic strategies warrant investigation:

• Small molecule inhibitors: Design of specific inhibitors targeting BIRC8's unique mechanism of BAX-mediated apoptosis inhibition could provide selective therapeutic options.

• Peptide mimetics: Development of peptides that mimic the binding interfaces between BIRC8 and its interaction partners could disrupt its anti-apoptotic function.

• Antisense oligonucleotides or siRNA: Given BIRC8's upregulation in various cancers , nucleic acid-based therapies targeting BIRC8 mRNA could be effective, particularly with advances in delivery technologies.

• Dual pathway inhibition: Combination approaches targeting both BIRC8 and the Wnt signaling pathway might yield synergistic effects in cancers where both pathways are active.

• Immunotherapy approaches: If BIRC8 expression is sufficiently cancer-specific, it could potentially serve as a target for CAR-T cell therapy or therapeutic antibodies, though extensive validation of its restricted expression pattern would be necessary.

These therapeutic approaches should be explored with careful consideration of tissue-specific expression patterns and potential off-target effects, as BIRC8 expression has been detected in normal testis and lymphoblastic cells .