BCL2L1 Antibody

What is BCL2L1 and why is it important in research?

BCL2L1 (Bcl-2-like protein 1) is a member of the BCL-2 protein family that regulates apoptosis. It exists in multiple isoforms, with the longer isoform (Bcl-XL) acting as an anti-apoptotic regulator and the shorter form (Bcl-xS) functioning as an apoptotic activator. BCL2L1 is critically important in research because it:

• Functions as a potent inhibitor of cell death by inhibiting caspase activation

• Regulates cell death by blocking the voltage-dependent anion channel (VDAC)

• Prevents the release of cytochrome C from mitochondrial membranes

• Acts as a regulator of G2 checkpoint and progression to cytokinesis during mitosis

• Is associated with drug resistance and disease progression in numerous cancers

Understanding BCL2L1's function is essential for research in apoptosis regulation, cancer biology, and developmental processes.

How do I choose between monoclonal and polyclonal BCL2L1 antibodies?

The choice between monoclonal and polyclonal BCL2L1 antibodies should be based on your specific research application:

Monoclonal Antibodies:

• Offer high specificity for a single epitope (e.g., mouse monoclonal antibody clone 804CT19.1.4)

• Provide consistent lot-to-lot reproducibility

• Ideal for applications requiring high specificity like Western blotting

• Available in various formats such as IgG1 isotype antibodies

Polyclonal Antibodies:

• Recognize multiple epitopes on the BCL2L1 protein

• Often provide stronger signals due to binding of multiple antibodies

• Better for detecting proteins with altered conformation or denatured proteins

• Examples include rabbit polyclonal antibodies that target specific amino acid regions (AA 1-219, AA 81-200, etc.)

For critical experiments, it's advisable to validate findings with both types of antibodies to ensure robust results.

What specific target regions should I consider when selecting a BCL2L1 antibody?

Different BCL2L1 antibodies target various regions of the protein, which affects their application suitability:

When selecting an antibody, consider:

• Which isoform you need to detect (Bcl-XL vs. Bcl-xS)

• Whether the epitope is accessible in your experimental conditions

• If post-translational modifications might interfere with antibody binding

What are the optimal conditions for using BCL2L1 antibodies in Western blotting?

For optimal Western blotting with BCL2L1 antibodies:

Sample Preparation:

• Use appropriate lysis buffers containing protease inhibitors

• The calculated molecular weight of BCL2L1 is approximately 26049 Da , but observed weight can be 26-30 kDa

Protocol Recommendations:

• Dilution: Generally use 1:1000 dilution for Western blotting

• Blocking: 5% non-fat milk or BSA in TBST is typically effective

• Detection: Both chemiluminescence and fluorescence detection are suitable

• Positive controls: Consider using cell lines known to express high levels of BCL2L1

Troubleshooting:

• If detecting BCL-XL specifically, ensure your antibody recognizes this isoform

• Multiple bands may represent different isoforms or post-translational modifications

• For weak signals, consider longer exposure times or higher antibody concentration

ABIN5518741 antibody has been validated for optimal WB results at 0.1-0.5 μg/mL concentration .

How can I optimize immunohistochemistry protocols for BCL2L1 detection in tissue samples?

For successful IHC detection of BCL2L1:

Tissue Preparation:

• Formalin-fixed, paraffin-embedded (FFPE) tissues require antigen retrieval

• Heat-mediated antigen retrieval in 10 mM citrate buffer (pH 6.0) for 20 minutes is recommended

Protocol Optimization:

• Concentration: Start with 0.5-1 μg/mL for paraffin sections

• Incubation: Overnight at 4°C often yields best results

• Detection systems: DAB chromogen provides good contrast for visualization

• Counterstaining: Hematoxylin works well for nuclear contrast

Important Considerations:

• Always include positive and negative controls

• Some antibodies (like CPTC-BCL2L1-1) have shown negative results in IHC applications , indicating not all BCL2L1 antibodies are suitable for IHC

• BCL2L1 may localize to multiple cellular compartments including mitochondria, cytoplasm, and nucleus

It's worth noting that the CPTC-BCL2L1-1 antibody has been evaluated as "Negative" for IHC applications by the Human Protein Atlas , highlighting the importance of selecting appropriate antibodies for this application.

How can I use protein-protein interaction assays to study BCL2L1 interactions?

BCL2L1 interacts with several proteins, particularly those in the BCL-2 family. Here are methodologies for studying these interactions:

Proximity Ligation Assay (PLA):

• Effective for detecting BCL2L1-BAX interactions in situ

• Use antibody pairs like BCL2L1 rabbit polyclonal antibody (1:1200) with BAX mouse monoclonal antibody (1:50)

• Each red dot in PLA represents a protein-protein interaction complex

• Analysis can be performed using software like BlobFinder from Uppsala University

Co-Immunoprecipitation (Co-IP):

• Use BCL2L1 antibodies to pull down protein complexes

• Western blot for interacting partners such as BAX, BAK, or BIM

• Consider cross-linking if interactions are transient

Surface Plasmon Resonance (SPR):

• The CPTC-BCL2L1-1 antibody has shown high binding in SPR assays

• Useful for quantitative measurement of binding kinetics

• Can determine association/dissociation constants for protein interactions

Remember that BCL2L1's interactions may be regulated by post-translational modifications, particularly phosphorylation, which has been observed at sites like Ser-49 .

What are common challenges in BCL2L1 antibody experiments and how can they be addressed?

Researchers often encounter several challenges when working with BCL2L1 antibodies:

Issue: Multiple Bands in Western Blots

• Cause: Detection of different isoforms (Bcl-XL ~26 kDa, Bcl-xS ~19 kDa)

• Solution: Use isoform-specific antibodies or reference the expected molecular weights

Issue: Weak or No Signal in IHC

• Cause: Inadequate antigen retrieval or epitope masking

• Solution: Optimize antigen retrieval methods; some BCL2L1 antibodies (e.g., CPTC-BCL2L1-1) consistently show negative results in IHC

Issue: Background Staining

• Cause: Non-specific binding or high antibody concentration

• Solution: Increase blocking time/concentration, reduce primary antibody concentration, or use monoclonal antibodies for higher specificity

Issue: Inconsistent Results Across Cell Types

• Cause: Differential expression of BCL2L1 isoforms in various tissues

• Solution: Validate antibody performance in your specific cell type; BCL2L1 expression varies significantly between tissues

A systematic validation approach using multiple detection methods and appropriate controls is recommended for conclusive results.

How can I validate the specificity of my BCL2L1 antibody?

Rigorous validation is critical for ensuring antibody specificity:

Knockout/Knockdown Controls:

• Use BCL2L1 knockout cells or siRNA knockdown

• Compare staining patterns before and after BCL2L1 depletion

• Quantify reduction in signal corresponding to knockdown efficiency

Multiple Antibody Validation:

• Use antibodies targeting different epitopes of BCL2L1

• Consistent results across different antibodies increase confidence

Peptide Competition Assay:

• Pre-incubate antibody with the immunizing peptide

• Specific signal should be abolished or significantly reduced

Recombinant Protein Control:

• Test antibody against purified His-tagged BCL2L1 protein

• Evaluate cross-reactivity with similar family members (BCL2, MCL1)

Immunoblotting Paired with Mass Spectrometry:

• Confirm the identity of the detected protein band

• Particularly important for novel applications or cell types

The Human Protein Atlas and NCI have conducted extensive validation of some BCL2L1 antibodies, providing valuable reference data .

How can BCL2L1 antibodies be utilized in studying apoptosis resistance in cancer?

BCL2L1 (Bcl-XL) is a critical anti-apoptotic protein implicated in cancer therapy resistance. Advanced research applications include:

Monitoring Therapy Response:

• Use BCL2L1 antibodies to track expression changes before and after treatment

• Correlate expression levels with patient outcomes and drug resistance

• BCL2L1 overexpression has been associated with resistance to various chemotherapeutics

Combination Therapy Studies:

• Assess BCL2L1 expression in response to BCL-XL inhibitors

• Investigate synergistic effects with other apoptosis-inducing therapies

• Evaluate potential compensatory upregulation of other anti-apoptotic proteins

Antibody-Drug Conjugates (ADCs):

• Recent research has developed BCL-XL-targeting ADCs that show promising results in preclinical models

• These conjugates overcome cardiovascular toxicity issues seen with small molecule inhibitors

• The EGFR-targeting ADC AM1-15 showed inhibition of tumor xenograft growth without cardiovascular toxicity

Spatial Distribution Analysis:

• Use immunofluorescence to examine subcellular localization in cancer cells

• BCL2L1 can localize to mitochondria, cytoplasm, and nucleus, affecting its function

• Changes in localization may indicate alterations in apoptotic pathways

What is the role of BCL2L1 in stem cell differentiation and how can antibodies help study this process?

BCL2L1 plays a crucial role in stem cell survival and differentiation:

Pancreatic Cell Differentiation:

• BCL-xL exhibits increased expression during pancreatic differentiation from pluripotent stem cells

• Western blotting with BCL2L1 antibodies showed reciprocal upregulation of BCL-xL and downregulation of BAK during differentiation

• This coincides with decreased cleaved CASP3 expression, suggesting BCL-xL promotes survival of differentiating pancreatic progenitors

Research Approaches:

• Temporal expression analysis: Track BCL2L1 levels during differentiation stages

• Knockdown studies: shRNA against BCL2L1 demonstrated that pancreatic progenitors experience higher cell death when BCL2L1 is depleted

• Inhibitor studies: Treatment with BCL-XL inhibitor WEHI-539 resulted in decreased BCL2L1 transcript levels and increased cell death

Key Findings:

• Later-stage differentiating pancreatic progenitors appear more reliant on BCL-xL for survival

• Upon loss of BCL-xL expression, less differentiated progenitors remain and express lower levels of pancreatic genes

• This suggests BCL-xL is essential for establishing both survival and identity of well-differentiated pancreatic progenitors

Immunostaining analyses can confirm upregulation of BCL-xL protein during differentiation, as demonstrated in studies showing increased expression from day 3 to day 12 of differentiation .

How can BCL2L1 phosphorylation be detected and what is its functional significance?

BCL2L1 function is regulated by post-translational modifications, particularly phosphorylation:

Detection Methods:

• Phospho-specific antibodies: Antibodies targeting pSer62-BCL2L1 are available for research

• Western blotting: Use phospho-specific antibodies followed by total BCL2L1 antibodies

• Mass spectrometry: For unbiased identification of all phosphorylation sites

• Phos-tag gels: Can separate phosphorylated from non-phosphorylated BCL2L1

Key Phosphorylation Sites:

• Ser-49: When phosphorylated, BCL2L1 localizes to the centrosome

• Ser-62: Commonly studied phosphorylation site with available antibodies

• Multiple sites have been described in T cells and B cells (serine 70, serine 87, and threonine 69)

Functional Significance:

• Affects subcellular localization: After neuronal stimulation, BCL2L1 translocates from cytosol to synaptic vesicle and mitochondrion membrane in a calmodulin-dependent manner

• Regulates protein-protein interactions: Phosphorylation can alter binding affinity for pro-apoptotic partners

• Modulates protein stability: Can affect the half-life of BCL2L1 protein

Understanding phosphorylation patterns is crucial for developing therapeutic strategies targeting BCL2L1 in diseases like cancer.

What are the latest developments in BCL2L1-targeting therapeutics and how can antibodies aid in their development?

Recent advances in BCL2L1-targeting therapeutics include:

Antibody-Drug Conjugates (ADCs):

• Novel BCL-XL-targeting ADCs show promising preclinical results

• Mirzotamab clezutoclax, containing the AAA drug-linker, is the first selective BCL-XL–targeting agent to enter human clinical trials

• BCL2L1 antibodies are essential for validating target engagement and mechanism of action

Overcoming Toxicity Challenges:

• Selective BCL-XL inhibitors showed severe cardiovascular toxicity in preclinical species

• Modified drug-linker technologies in ADCs have mitigated these toxicities

• BCL2L1 antibodies help identify tissue-specific expression patterns to predict and understand toxicities

Combination Approaches:

• BCL2L1 inhibition combined with other targeted therapies shows synergistic effects

• Understanding resistance mechanisms through antibody-based detection of compensatory pathways

• Patient stratification based on BCL2L1 expression levels detected by immunohistochemistry

Target Validation Methodologies:

• Confirmation of BCL2L1 expression in tumor samples using validated antibodies

• Correlation of expression levels with clinical outcomes

• Monitoring of on-target effects in clinical trial samples

The advancement of mirzotamab clezutoclax to clinical trials represents a significant milestone in BCL2L1-targeted therapeutics and provides opportunities for further development in this field .

How can multi-parameter analysis with BCL2L1 antibodies enhance our understanding of apoptotic regulation?

Modern multi-parameter approaches provide deeper insights into BCL2L1 function:

Multiplexed Immunofluorescence:

• Simultaneous detection of BCL2L1 with other BCL-2 family members

• Assessment of co-localization patterns with mitochondrial markers

• Quantitative image analysis to determine protein expression ratios

Single-Cell Analysis:

• Flow cytometry with BCL2L1 antibodies combined with apoptosis markers

• Single-cell RNA-seq paired with protein detection for correlation of transcript and protein levels

• Mass cytometry (CyTOF) for high-dimensional analysis of BCL2L1 in cellular contexts

Proximity Ligation Assays (PLA):

• Detection of specific protein-protein interactions between BCL2L1 and binding partners

• BCL2L1 & BAX Protein Interaction Antibody Pairs allow visualization of interaction complexes in situ

• Each red dot in PLA represents detection of a protein-protein interaction complex

Spatial Transcriptomics:

• Combining BCL2L1 antibody staining with spatial transcriptomic approaches

• Understanding regional variation in expression within tissues

• Correlation with markers of cellular stress or damage

These multi-parameter approaches are particularly valuable for understanding the complex regulatory networks governing apoptosis and can reveal context-specific functions of BCL2L1.

What are the considerations for using BCL2L1 antibodies in clinical diagnostic applications?

While BCL2L1 antibodies are primarily research tools, their potential in clinical diagnostics warrants consideration:

Standardization Requirements:

• Rigorous validation across multiple tissue types and conditions

• Reproducibility across different laboratories and platforms

• Establishment of scoring systems and cutoff values for interpretation

Potential Diagnostic Applications:

• Prognostic marker in various cancers

• Predictive biomarker for response to BCL-XL inhibitors or other apoptosis-targeting therapies

• Monitoring treatment response over time

Technical Considerations:

• Automated staining platforms for consistent results

• Digital pathology for quantitative assessment

• Quality control measures including appropriate positive and negative controls

Regulatory Aspects:

• IVD (In Vitro Diagnostic) certification requirements

• Compliance with laboratory developed test (LDT) regulations

• Inclusion in clinical guidelines and diagnostic algorithms

Important Caveats:

• Most current BCL2L1 antibodies are labeled "for research use only and not for use in diagnostic or therapeutic procedures"

• CPTC-BCL2L1-1 showed negative results in IHC evaluations by the Human Protein Atlas

• Careful antibody selection based on validation in specific contexts is essential