BCL2A1 Antibody

What is BCL2A1 and what cellular functions does it regulate?

BCL2A1 (BCL2-related protein A1) is a member of the BCL-2 protein family that acts primarily as an anti-apoptotic regulator. It forms hetero- or homodimers with other BCL-2 family proteins and plays a crucial role in regulating programmed cell death. With a molecular weight of approximately 20kDa, BCL2A1 is primarily localized in the cytoplasm where it contributes to cell survival mechanisms. The protein is involved in various cellular activities including embryonic development, tissue homeostasis, and has been implicated in tumorigenesis . BCL2A1 functions within the A1/Bim axis, which is targeted by key kinases mediating B-cell receptor (BCR)-dependent survival signals, such as spleen tyrosine kinase (Syk) and Brutons tyrosine kinase (Btk) .

What are the common applications for BCL2A1 antibodies in research?

BCL2A1 antibodies serve as essential tools in multiple research applications:

• Western Blot (WB): Most commonly used at dilutions between 1:500-1:2000 to detect BCL2A1 protein expression in cell and tissue lysates .

• Immunofluorescence/Immunocytochemistry (IF/ICC): Typically used at dilutions of 1:50-1:200 to visualize BCL2A1 localization within cells .

• ELISA: For quantitative measurement of BCL2A1 protein levels .

• Mechanistic studies: Investigating apoptotic pathways, particularly in cancer and immune cell research .

• Drug development research: Especially for therapies targeting cell survival pathways in cancer .

These applications allow researchers to investigate BCL2A1's role in normal physiology and disease states, particularly in cancer biology, immunology, and drug development contexts.

How do I select the appropriate BCL2A1 antibody for my specific experiment?

Selection of an appropriate BCL2A1 antibody depends on several factors:

• Research application: Different antibodies are optimized for specific applications. For instance, CAB0134 is validated for WB, IF/ICC, and ELISA applications , while 12223-1-AP is primarily cited for WB and ELISA .

• Species reactivity: Confirm the antibody reacts with your species of interest. For example, both CAB0134 and 12223-1-AP are reactive with human samples .

• Isotype and host species: Consider compatibility with your detection systems. Many BCL2A1 antibodies, like CAB0134 and 12223-1-AP, are rabbit IgG polyclonal antibodies .

• Epitope recognition: Review the immunogen information. For example, CAB0134 targets a synthetic peptide within amino acids 70-150 of human BCL2A1 .

• Validation data: Look for antibodies with published citations demonstrating successful use in your application of interest .

When selecting between polyclonal and monoclonal antibodies, consider that polyclonals offer broader epitope recognition but potentially lower specificity, while monoclonals provide higher specificity but might have lower sensitivity for detecting proteins with conformational changes.

How can BCL2A1 antibodies be used to study resistance mechanisms in cancer therapy?

BCL2A1 antibodies serve as critical tools for investigating therapy resistance mechanisms in cancer, particularly in B-cell lymphomas. Recent research has revealed that BCL2A1 upregulation contributes significantly to resistance against targeted therapies:

• Mechanistic studies: BCL2A1 antibodies can detect increased BCL2A1 expression associated with CARD11 gain-of-function mutations, which has been identified as a mechanism of resistance to targeted therapies in mantle cell lymphoma (MCL) .

• Resistance biomarker detection: Immunoblotting with BCL2A1 antibodies can help identify tumors likely to develop resistance to BCL2 inhibitors like venetoclax, as concurrent upregulation of BCL-XL and BCL2A1 induces resistance to such therapies .

• Therapeutic response monitoring: Sequential tumor biopsies analyzed with BCL2A1 antibodies can track changes in BCL2A1 expression during treatment, potentially predicting emergence of resistant subclones .

• Pathway analysis: BCL2A1 antibodies enable investigation of the CARD11/NF-κB1/BCL2A1 axis, which has been identified as a key pathway in therapy resistance that can be counteracted by MALT1/BCL2 dual targeting strategies .

This research has significant clinical implications, as demonstrated by the OAsIs clinical trial (NCT02558816), which revealed that BCL2A1 upregulation via NF-κB1 activation represents a common escape mechanism from targeted therapies in aggressive B-cell lymphomas .

What are the current methods for studying BCL2A1 protein interactions using antibody-based approaches?

Several antibody-based approaches are employed to study BCL2A1 protein interactions:

• Co-immunoprecipitation (Co-IP): Using BCL2A1 antibodies to pull down protein complexes and identify interaction partners. This technique has helped elucidate BCL2A1's interactions with other BCL-2 family members and regulatory proteins.

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ, providing spatial information about BCL2A1 interactions within cells.

• Immunofluorescence co-localization: Using BCL2A1 antibodies in combination with antibodies against potential interaction partners to assess co-localization in cellular compartments.

• Chromatin Immunoprecipitation (ChIP): For studying transcription factors that regulate BCL2A1 expression, such as NF-κB factors that are activated downstream of CARD11 .

• BiFC (Bimolecular Fluorescence Complementation): When combined with epitope tagging, BCL2A1 antibodies can validate protein interactions observed through BiFC assays.

These methods have revealed that BCL2A1 interacts with various proteins in the A1/Bim axis, which is targeted by key kinases mediating B-cell receptor (BCR)-dependent survival signals . Furthermore, they have demonstrated how CARD11 gain-of-function mutations lead to BCR-independent overexpression of NF-κB1 target genes, including BCL2A1 .

What are the most common technical challenges when using BCL2A1 antibodies in Western blot, and how can they be addressed?

Several challenges may arise when using BCL2A1 antibodies in Western blot applications:

• Non-specific binding:

  • Problem: Multiple bands appearing on blots.

  • Solution: Optimize antibody dilution (try the recommended range of 1:500-1:2000) . Increase blocking time and concentration, and include 0.1% Tween-20 in washing buffer.

• Weak or no signal:

  • Problem: Insufficient protein or antibody concentration.

  • Solution: Ensure adequate protein loading (30-50μg total protein), optimize transfer conditions, and consider longer primary antibody incubation at 4°C overnight.

• Variable molecular weight detection:

  • Problem: BCL2A1 may appear at different sizes than its calculated MW of 20kDa due to post-translational modifications.

  • Solution: Reference the manufacturer's observed MW data . Consider using positive control lysates with confirmed BCL2A1 expression.

• Antibody specificity issues:

  • Problem: Cross-reactivity with BCL2A1 homologs or isoforms.

  • Solution: Validate results using multiple antibodies targeting different epitopes. For example, CAB0134 targets amino acids 70-150 , while other antibodies may target different regions.

• Sample preparation challenges:

  • Problem: Protein degradation during extraction.

  • Solution: Include protease inhibitors in lysis buffers, keep samples cold, and minimize freeze-thaw cycles.

For optimal results, follow the manufacturer's recommended protocols and consider that storage conditions (e.g., -20°C storage with 50% glycerol for CAB0134) can significantly impact antibody performance.

How do I design experiments to assess BCL2A1 expression changes in response to therapeutic interventions?

Designing robust experiments to assess BCL2A1 expression changes requires careful planning:

• Baseline establishment:

  • Measure BCL2A1 expression in treatment-naïve cells/tissues using quantitative methods (qPCR, Western blot with BCL2A1 antibodies)

  • Include appropriate control cell lines with known BCL2A1 expression levels

• Time-course analysis:

  • Monitor BCL2A1 expression at multiple timepoints after treatment (early: 6-24h; intermediate: 48-72h; late: >96h)

  • Use consistent protein loading amounts and normalize to housekeeping proteins

• Dose-response relationship:

  • Test multiple drug concentrations to establish dose-dependent effects on BCL2A1 expression

  • Include sub-therapeutic doses to identify compensatory upregulation mechanisms

• Combination studies:

  • When testing drug combinations, use both single agents and combinations to identify synergistic effects

  • This approach helped identify that MALT1 inhibition combined with BCL2 inhibition induces synergistic cell death regardless of CARD11 mutational status

• Multiple detection methods:

  • Validate findings using complementary techniques:

    • Protein level: Western blot with BCL2A1 antibodies (1:500-1:2000 dilution)

    • mRNA level: qRT-PCR

    • In situ detection: Immunofluorescence (1:50-1:200 dilution)

• Functional correlation:

  • Parallel assessment of cell viability, apoptosis markers, and BCL2A1-dependent pathways

  • Include known BCL2A1 regulatory pathway components (e.g., NF-κB1, CARD11)

This comprehensive approach was successfully employed in studies demonstrating that CARD11 gain-of-function mutations lead to BCL2A1 upregulation and therapy resistance in B-cell lymphomas .

How are BCL2A1 antibodies being utilized in single-cell analysis methods?

BCL2A1 antibodies are increasingly being integrated into single-cell analysis methods, opening new research avenues:

• Single-cell proteomics:

  • Mass cytometry (CyTOF) incorporating BCL2A1 antibodies allows simultaneous detection of BCL2A1 with dozens of other proteins at single-cell resolution

  • This reveals heterogeneity in BCL2A1 expression within seemingly homogeneous populations

• Single-cell RNA-sequencing validation:

  • BCL2A1 antibodies validate protein-level correlates of transcriptomic findings

  • Recent studies used this approach to identify BCR-independent overexpression of NF-κB1 target genes, including BCL2A1, in treatment-resistant MCL cells

• Spatial transcriptomics correlation:

  • Combining BCL2A1 immunohistochemistry with spatial transcriptomics provides insights into microenvironmental regulation of BCL2A1 expression

• Microfluidic single-cell isolation:

  • BCL2A1 antibodies can be used to identify and isolate specific cell populations using microfluidic technologies based on microdroplets and valve systems

  • This approach requires small input material, has low process cost, high speed, and precise control

• Computational analysis integration:

  • Machine learning algorithms can integrate single-cell BCL2A1 expression data with other parameters to predict therapy response

  • This approach helped develop a 16-gene resistance signature for MCL patients that predicted response to both targeted therapies and conventional chemotherapy

These emerging applications highlight how BCL2A1 antibodies contribute to understanding cellular heterogeneity and identifying resistant subpopulations that might drive treatment failure.

What is the role of BCL2A1 in immune cell function, and how can antibodies help elucidate these mechanisms?

BCL2A1 plays diverse roles in immune cell function, which can be effectively studied using antibody-based approaches:

• B-cell homeostasis regulation:

  • BCL2A1 antibodies have revealed that conditional knockdown of BCL2A1 protein expression significantly impacts mature B-cell homeostasis

  • The protein acts downstream of the B-cell receptor (BCR) signaling pathway, mediating survival signals from key kinases including Syk and Btk

• Myeloid cell development and function:

  • Research using constitutive knockdown models indicated possible roles for BCL2A1 in myeloid compartment development and homeostasis

  • BCL2A1 antibodies can track expression changes during myeloid differentiation and activation

• Lymphocyte development:

  • Antibody-based studies have demonstrated rate-limiting roles for BCL2A1 in lymphocyte development and granulopoiesis

  • Western blot analysis using appropriate dilutions (1:500-1:2000) can quantify BCL2A1 expression at different developmental stages

• Mast cell activation:

  • BCL2A1 has been implicated in mast cell activation processes

  • Immunofluorescence using BCL2A1 antibodies (1:50-1:200 dilution) can visualize BCL2A1 localization during activation events

• T-cell responses:

  • Emerging research suggests BCL2A1 may regulate T-cell survival during immune responses

  • Antibody-based flow cytometry can identify BCL2A1-expressing T-cell subsets

Understanding these immunological functions has significant implications for developing therapies targeting autoimmune diseases and enhancing anti-tumor immunity. For instance, the identification of the CARD11/NF-κB1/BCL2A1 axis as a resistance mechanism in B-cell lymphomas suggests potential therapeutic approaches counteracting this pathway .

How can researchers investigate the impact of post-translational modifications on BCL2A1 using antibody-based techniques?

Investigating post-translational modifications (PTMs) of BCL2A1 requires specialized antibody-based approaches:

• Modification-specific antibodies:

  • Researchers can develop or acquire antibodies specifically targeting known BCL2A1 PTMs (phosphorylation, ubiquitination, acetylation)

  • These antibodies should be validated through positive controls using cells treated with PTM-inducing agents

• Two-dimensional gel electrophoresis:

  • Combining 2D-PAGE with Western blotting using BCL2A1 antibodies can separate protein isoforms with different PTMs

  • This technique helps identify shifts in isoelectric point or molecular weight indicative of modifications

• Immunoprecipitation followed by mass spectrometry:

  • Using BCL2A1 antibodies (like CAB0134 or 12223-1-AP) to immunoprecipitate the protein

  • Subsequent mass spectrometry analysis identifies specific modifications and their sites

  • The sequence information provided in product specifications (amino acids 70-150 for CAB0134) helps interpret mass spectrometry data

• Functional correlation studies:

  • Compare BCL2A1 modification states in normal versus disease states (e.g., between therapy-responsive and therapy-resistant lymphomas)

  • Correlate modifications with BCL2A1 stability, localization, and anti-apoptotic function

• PTM enzyme manipulation:

  • Overexpress or inhibit enzymes responsible for BCL2A1 modifications

  • Monitor effects using Western blot (1:500-1:2000 dilution) or immunofluorescence (1:50-1:200 dilution)

Understanding BCL2A1 PTMs has significant implications for therapy development, as modifications may influence protein stability and function. This is particularly relevant in the context of therapy resistance in B-cell lymphomas, where BCL2A1 upregulation promotes resistance to targeted therapies .