BCL2L11 Antibody, Biotin conjugated

What is BCL2L11 and what role does it play in cellular processes?

BCL2L11 (BCL2-like protein 11), also known as Bim, is a pro-apoptotic member of the BCL2 protein family that plays a critical role in programmed cell death. The protein functions as a mediator of cell death and is essential for normal immune system development and homeostasis. BCL2L11 initiates apoptosis by interacting with and neutralizing anti-apoptotic BCL2 family members, thereby facilitating the activation of BAX and BAK, which ultimately leads to mitochondrial outer membrane permeabilization and cell death. In research contexts, BCL2L11 has been extensively studied for its role in cancer development, particularly in lymphomas where its silencing contributes to lymphomagenesis by preventing MYC-driven apoptosis . The protein has a calculated molecular weight of approximately 22 kDa but is typically observed as bands of 23 and 18 kDa in Western blot applications due to post-translational modifications and different isoforms .

How should BCL2L11 Antibody, Biotin conjugated be stored and handled to maintain optimal activity?

For optimal stability and activity, BCL2L11 Antibody, Biotin conjugated should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can degrade the antibody and reduce its effectiveness . For short-term storage (up to three months), the antibody can be kept at 4°C, but for long-term storage (up to one year), -20°C is recommended .

What are the typical applications for BCL2L11 Antibody, Biotin conjugated in laboratory research?

BCL2L11 Antibody, Biotin conjugated is utilized in various research applications, with ELISA being the primary application noted in the product information . The biotin conjugation provides advantages for detection systems that utilize streptavidin, which has an exceptionally high affinity for biotin. This high-affinity interaction enables sensitive detection in various experimental setups.

While the biotin-conjugated version is specifically indicated for ELISA applications, non-conjugated BCL2L11 antibodies have broader application ranges including Western Blot (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), and Immunofluorescence (IF) . When adapting protocols for the biotin-conjugated version, researchers should consider the following methodological adjustments:

It's important to note that each experimental system may require optimization of antibody dilutions and conditions to obtain optimal results .

What species reactivity can be expected with BCL2L11 Antibody, Biotin conjugated?

Based on the provided search results, the BCL2L11 Antibody, Biotin conjugated from AFG Scientific is specifically designed to react with human BCL2L11 protein . The immunogen used for antibody generation was recombinant Human Bcl-2-like protein 11 (amino acids 1-198) , which enhances its specificity for human samples.

Other BCL2L11 antibodies available from different manufacturers may have broader species reactivity. For instance, the non-conjugated BCL2L11 antibody from Proteintech (product 22037-1-AP) shows reactivity with both human and mouse samples , while the antibody from Boster Bio (A01552-2) reacts with human, mouse, and rat species .

When selecting an antibody for your research, it's crucial to match the species reactivity with your experimental model. Cross-reactivity between species may occur due to conserved epitopes in the BCL2L11 protein sequence across mammals, but this should be experimentally validated for your specific application and sample type.

How can BCL2L11 Antibody, Biotin conjugated be used to study BCL2L11's role in virus-induced lymphomagenesis?

BCL2L11 Antibody, Biotin conjugated can serve as a powerful tool for investigating the mechanisms by which oncogenic viruses like Epstein-Barr virus (EBV) silence BCL2L11 expression to promote lymphomagenesis. Research has shown that EBV-encoded proteins EBNA3A and EBNA3C inactivate a BCL2L11 enhancer-promoter hub through the recruitment of Polycomb Repressive Complex 2 (PRC2) . This silencing of BCL2L11 prevents apoptosis that would normally be triggered by MYC activation, contributing to lymphoma development.

To study this process, researchers can design experiments using the following methodological approach:

• Chromatin Immunoprecipitation (ChIP) assays: Use BCL2L11 Antibody, Biotin conjugated in ChIP experiments to assess the binding of EBV proteins (EBNA3A, EBNA3C) to the BCL2L11 locus in infected versus uninfected cells. The biotin conjugation facilitates pull-down with streptavidin beads.

• Chromosome Conformation Capture (3C) techniques: Combine with immunoprecipitation using the BCL2L11 antibody to investigate changes in enhancer-promoter interactions at the BCL2L11 locus following EBV infection.

• Comparative expression analysis: Quantify BCL2L11 protein levels in various EBV-positive and negative lymphoma cell lines using the antibody in Western blot or flow cytometry applications.

• Drug intervention studies: Examine how epigenetic drugs that target PRC2 activity affect BCL2L11 expression in EBV-positive cells, potentially reversing the silencing and inducing apoptosis .

This research approach could provide valuable insights into how viruses manipulate host cell gene expression to promote oncogenesis and might identify potential therapeutic targets for virus-associated lymphomas.

What technical challenges might arise when using BCL2L11 Antibody, Biotin conjugated in proximity biotinylation (BioID) experiments, and how can they be addressed?

Proximity biotinylation (BioID) is a powerful technique to identify protein-protein interactions in living cells. When combining this approach with BCL2L11 Antibody, Biotin conjugated, several technical challenges may arise:

Challenge 1: Background from endogenous biotinylated proteins
Cells contain endogenously biotinylated carboxylases that appear as background in streptavidin pull-downs .

Solution: Include appropriate controls in your experimental design. As demonstrated in the BioID experiments with tBid-BirA*, compare results with BirA* alone to distinguish specific interactions from background . Additionally, pre-clear lysates with avidin beads before immunoprecipitation with BCL2L11 Antibody, Biotin conjugated.

Challenge 2: Pro-apoptotic activity of BCL2L11 affecting experimental outcomes
Similar to the challenge faced with tBid-BirA* fusion proteins, the pro-apoptotic nature of BCL2L11 can lead to cell death during experiments, resulting in weaker biotinylation signals .

Solution: Use inducible expression systems to control the timing and level of BCL2L11 expression. Alternatively, employ anti-apoptotic inhibitors or use BCL2L11 mutants with reduced apoptotic activity while maintaining interaction capabilities.

Challenge 3: Distinguishing between direct and indirect interactions
BioID identifies proteins in proximity, but doesn't distinguish direct binding partners from nearby proteins.

Solution: Combine BioID results with complementary approaches such as co-immunoprecipitation using BCL2L11 Antibody, Biotin conjugated or in vitro binding assays to validate direct interactions.

Challenge 4: Interference from the biotin conjugate
Having both a biotin-conjugated antibody and biotin-labeled proteins from BioID can create interpretation challenges.

Solution: Consider using sequential purification approaches:

• First, isolate BioID-labeled proteins with streptavidin beads

• Then perform immunoprecipitation with non-biotinylated BCL2L11 antibody

• Alternatively, use epitope-tagged BCL2L11 constructs for BioID experiments

How do different isoforms of BCL2L11 affect antibody recognition, and what methodological approaches can address isoform-specific detection?

BCL2L11 exists in multiple isoforms due to alternative splicing, with major isoforms including BCL2L11-EL (extra long), BCL2L11-L (long), and BCL2L11-S (short). These isoforms have different functional properties and expression patterns, which can significantly impact research findings.

The BCL2L11 Antibody, Biotin conjugated product is generated using recombinant Human Bcl-2-like protein 11 (amino acids 1-198) as the immunogen . This region may include epitopes present in multiple isoforms, potentially leading to recognition of several BCL2L11 variants. This is consistent with observations that BCL2L11 antibodies often detect bands at different molecular weights (typically 23 and 18 kDa) in Western blot applications .

Methodological approaches for isoform-specific detection:

• Epitope mapping: Determine which epitope(s) the antibody recognizes and cross-reference with isoform sequences to predict which variants will be detected.

• Validation with recombinant isoforms: Express individual BCL2L11 isoforms in a heterologous system and test antibody reactivity against each.

• Isoform-specific antibodies: When available, use antibodies raised against unique regions of specific isoforms.

• RT-PCR validation: Complement protein detection with RT-PCR using isoform-specific primers to confirm the expression pattern at the mRNA level.

• Mass spectrometry analysis: Perform mass spectrometry on immunoprecipitated samples to definitively identify which isoforms are present.

What are the experimental considerations when investigating BCL2L11-mediated apoptosis in relation to epigenetic regulation of enhancer-promoter interactions?

Investigating BCL2L11-mediated apoptosis in the context of epigenetic regulation requires careful experimental design to capture the complex interplay between chromatin structure, transcriptional regulation, and protein function. Based on the research findings regarding EBV-mediated silencing of BCL2L11 , several key experimental considerations emerge:

• Chromatin landscape analysis:

  • Employ ChIP-seq using antibodies against histone modifications (H3K27me3 for repression, H3K27ac for active enhancers) to map the epigenetic state of the BCL2L11 locus

  • Use ATAC-seq to identify accessible chromatin regions that may function as enhancers

  • Consider Hi-C or 4C-seq approaches to map the three-dimensional interactions between the BCL2L11 promoter and distal regulatory elements

• Enhancer-promoter interaction dynamics:

  • Utilize Chromosome Conformation Capture (3C) techniques to quantify specific interactions between the BCL2L11 promoter and identified enhancers under different conditions

  • Complement with live-cell imaging approaches using fluorescently tagged chromatin regions to visualize interaction dynamics

  • Investigate the role of chromatin remodelers like SWI/SNF (which affects MYC enhancer-promoter interactions) in BCL2L11 regulation

• Epigenetic perturbation approaches:

  • Apply epigenetic inhibitors targeting specific modifiers (e.g., EZH2 inhibitors to target PRC2 activity)

  • Use CRISPR-based approaches to specifically modify enhancer regions or to recruit epigenetic modifiers to the BCL2L11 locus

  • Monitor changes in BCL2L11 expression and apoptosis following epigenetic perturbations

• Functional readouts:

  • Employ multiple apoptosis assays (Annexin V staining, caspase activation, TUNEL) to comprehensively assess the functional consequence of BCL2L11 regulation

  • Use BCL2L11 Antibody, Biotin conjugated in proximity ligation assays to investigate how epigenetic changes affect BCL2L11 interactions with other proteins

• Integration of multi-omics data:

  • Combine ChIP-seq, RNA-seq, and proteomics data to build comprehensive models of BCL2L11 regulation

  • Apply systems biology approaches to identify key nodes in the regulatory network that may serve as therapeutic targets

By systematically addressing these experimental considerations, researchers can gain deeper insights into how epigenetic mechanisms regulate BCL2L11-mediated apoptosis, potentially revealing new therapeutic avenues for diseases characterized by dysregulated apoptosis.