BCL2L14 Antibody

What is BCL2L14 and what are its key biological functions?

BCL2L14 (also known as BCL-G) is a non-typical member of the BCL-2 protein family that plays a significant role in apoptosis regulation. The human canonical protein consists of 327 amino acid residues with a molecular weight of approximately 36.6 kDa and is primarily localized in the cytoplasm . Alternative splicing produces three different isoforms, with the long isoform (BCL-G L) and short isoform (BCL-G S) being the most studied . BCL-G functions as an apoptosis facilitator, suggesting potential tumor suppressor activity. Its expression pattern is tissue-specific, with predominant expression in the gastrointestinal tract during early human fetal development, while showing low or undetectable levels in fetal adrenal glands, heart, kidney, and lungs .

What types of BCL2L14 antibodies are currently available for research?

Researchers have access to a diverse range of BCL2L14 antibodies, including:

• Polyclonal antibodies: Derived from multiple B-cell lineages, these recognize various epitopes and are suitable for multiple applications

• Monoclonal antibodies: Generated from single B-cell clones, offering higher specificity and consistency between batches

• Recombinant monoclonal antibodies: Produced through recombinant DNA technology, providing enhanced batch-to-batch reproducibility

Available antibodies differ in host species (rabbit being the most common), applications supported (WB, IHC, IF, ELISA, FC), and species reactivity (human, mouse, rat) .

What is the tissue expression profile of BCL2L14 and why is this important for antibody selection?

The tissue distribution of BCL2L14 shows a distinctive pattern that researchers should consider when planning experiments:

What are the optimal conditions for Western blot detection of BCL2L14?

For optimal Western blot detection of BCL2L14, researchers should consider the following protocol parameters:

When troubleshooting, be aware that observed band sizes may vary from the predicted 37 kDa to approximately 40 kDa due to post-translational modifications or detection of specific isoforms . For reproducible results, it's advisable to use tissue samples with known high expression such as testis or lung tissue lysates as positive controls.

What are the critical steps for successful immunohistochemical detection of BCL2L14?

Successful immunohistochemical detection of BCL2L14 requires careful attention to several key parameters:

• Antigen retrieval: Heat-mediated antigen retrieval with Tris/EDTA buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may be used as an alternative depending on the tissue type

• Antibody dilution: Most commercial antibodies perform optimally at dilutions between 1:20-1:200 for IHC applications

• Tissue selection: Human prostate cancer tissue has been validated for IHC detection, while in rodent models, kidney tubules and colon glandular epithelium show reliable BCL2L14 staining patterns

• Signal detection: For DAB-based visualization, an HRP-conjugated secondary antibody at 1:500 dilution typically provides good results

• Controls: Include a negative control by substituting the primary antibody with PBS while maintaining all other protocol steps to distinguish specific from non-specific staining

For cytoplasmic staining assessment, particular attention should be paid to mouse kidney tubules and rat colon glandular epithelium, which show characteristic BCL2L14 localization patterns .

How can I optimize immunofluorescence protocols for BCL2L14 detection?

Optimizing immunofluorescence for BCL2L14 requires methodical adjustment of several parameters:

• Cell/tissue preparation: Paraformaldehyde fixation (2%) provides good preservation of BCL2L14 epitopes while maintaining cellular architecture

• Antibody dilution: For immunofluorescence, optimal dilutions typically range from 1:50-1:500 depending on the specific antibody

• Cell types: MCF-7 cells and HeLa cells have been validated for successful IF detection of BCL2L14

• Counterstains: DAPI for nuclear staining helps contextualize the subcellular localization of BCL2L14

• Signal amplification: For weak signals, consider using tyramide signal amplification systems, which can increase sensitivity without increasing background

When assessing results, the cytoplasmic distribution pattern of BCL2L14 staining should be carefully evaluated, as nuclear staining may indicate non-specific antibody binding .

How can I validate the specificity of BCL2L14 antibodies for my experimental system?

Validating antibody specificity is critical for generating reliable data. For BCL2L14 antibodies, consider these validation approaches:

• Orthogonal validation: Compare protein detection with RNA expression data from RNAseq experiments

• Recombinant expression: Test antibody against cells with known overexpression of BCL2L14 versus controls

• siRNA knockdown: Assess antibody signal reduction in cells where BCL2L14 has been knocked down

• Immunoprecipitation: Perform IP followed by mass spectrometry to confirm the identity of the pulled-down protein

• Multi-antibody approach: Use different antibodies targeting distinct epitopes of BCL2L14 to confirm consistent detection patterns

Data from the Human Protein Atlas project has already employed orthogonal RNAseq validation and recombinant expression approaches for several commercial BCL2L14 antibodies, providing a valuable starting point for researchers selecting reagents .

What are the challenges in detecting specific BCL2L14 isoforms and how can they be overcome?

Detecting specific BCL2L14 isoforms presents several challenges due to their structural similarities:

• Isoform-specific regions: The long isoform (BCL-G L) and short isoform (BCL-G S) share significant sequence homology, making selective detection difficult

• Spatial distribution differences: Different isoforms may have distinct subcellular localizations or tissue distribution patterns

• Post-translational modifications: Phosphorylation events can affect antibody recognition and apparent molecular weight

To overcome these challenges:

• Use isoform-specific antibodies generated against unique regions of each variant

• Employ RT-PCR alongside protein detection to confirm isoform expression

• Consider using knockout/knockdown models to validate isoform-specific detection

• Perform subcellular fractionation before Western blotting to better distinguish isoforms based on their localization patterns

Research indicates that the short and long BCL-G isoforms may have distinct functions, with differential regulation by factors such as IFN-γ and TNF-α in colonic epithelial cells, making isoform-specific detection particularly important for functional studies .

What are potential sources of inconsistent BCL2L14 antibody performance and their solutions?

Inconsistent antibody performance can significantly impact research outcomes. For BCL2L14 antibodies, common issues include:

For reproducible results with BCL2L14 antibodies, proper storage at -20°C in aliquots containing 0.02% sodium azide and 50% glycerol at pH 7.3 is recommended to maintain antibody integrity over time .

What is the significance of BCL2L14 in cancer research and how can antibodies aid in these investigations?

BCL2L14's role in cancer biology presents several important research avenues:

• Chromosomal deletions: The BCL2L14-containing region of chromosome 12 is commonly deleted in pre-B acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), suggesting a potential tumor suppressor role

• Prostate cancer: Allelic losses within chromosome 12 have been reported in prostate cancer patients, and BCL2L14 antibodies have been validated for detection in prostate cancer tissue

• Apoptotic regulation: As an apoptosis facilitator, BCL2L14 may influence cancer cell survival and resistance to therapy

BCL2L14 antibodies can support cancer research through:

• Assessing expression patterns across cancer types and stages

• Evaluating correlations between expression levels and clinical outcomes

• Studying protein interactions with other apoptotic regulators

• Investigating the effects of genetic alterations on protein expression and function

Researchers should consider using multiple detection methods (IHC, WB, IF) to comprehensively characterize BCL2L14 expression and localization in cancer contexts .

How is BCL2L14 expression regulated and what experimental approaches can investigate this regulation?

BCL2L14 expression is subject to complex regulatory mechanisms that can be studied through various experimental approaches:

• Transcriptional regulation:

  • PAR bZIP proteins control BCL2L14 expression, particularly the BCL-G S isoform in human embryonic kidney cells

  • The human BCL2L14 promoter is activated by the thyrotroph embryonic factor (TEF) and suppressed by NFIL3

  • A functional intronic p53-binding site influences expression

• Cytokine-mediated regulation:

  • IFN-α and IFN-γ increase BCL-G expression in an IRF-1 and STAT1-dependent manner

  • IFN-γ and TNF-α synergistically upregulate BCL-G isoforms in colonic epithelial cells through STAT1, p65/NF-κB, BRM, and BRG1

Experimental approaches to study these regulatory mechanisms include:

• Promoter-reporter assays to investigate transcription factor binding

• ChIP assays to confirm protein-DNA interactions

• Cytokine stimulation experiments with Western blot or qRT-PCR readouts

• Mutation analysis of key regulatory sites

These studies can help elucidate the context-specific regulation of BCL2L14, which appears to vary between cell types and physiological conditions .

What are the current limitations in BCL2L14 antibody research and emerging methodologies to address them?

Current limitations in BCL2L14 antibody research and emerging solutions include:

• Isoform specificity challenges:

  • Traditional antibodies may not reliably distinguish between BCL-G isoforms

  • Solution: Development of recombinant antibodies with enhanced epitope specificity

• Post-translational modification detection:

  • Standard antibodies may miss or be affected by phosphorylation events

  • Solution: Phospho-specific antibodies for studying regulatory mechanisms

• Quantification limitations:

  • Semi-quantitative nature of traditional Western blotting

  • Solution: Digital Western blot platforms and advanced image analysis

• Spatial resolution:

  • Traditional IHC lacks subcellular resolution

  • Solution: Super-resolution imaging techniques combined with highly specific antibodies

• Temporal dynamics:

  • Static measurements miss dynamic changes in expression/localization

  • Solution: Live-cell imaging with tagged antibody fragments or biosensors

Emerging methodologies to advance BCL2L14 research include multiplex immunofluorescence for co-expression studies, proximity ligation assays for protein interaction analysis, and mass cytometry for high-dimensional single-cell protein profiling .

What are the best practices for storage and handling of BCL2L14 antibodies to maintain optimal performance?

To maintain optimal performance of BCL2L14 antibodies, researchers should follow these best practices:

• Storage conditions:

  • Store at -20°C in a non-frost-free freezer

  • Most commercial BCL2L14 antibodies are stable for one year after shipment when properly stored

  • For small volumes (20µl), aliquoting may be unnecessary for -20°C storage

• Buffer composition:

  • Optimal storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some preparations may contain 0.1% BSA as a stabilizer

• Handling protocols:

  • Avoid repeated freeze-thaw cycles

  • Thaw completely before use and mix gently (do not vortex)

  • Keep on ice when in use

  • Return to -20°C promptly after use

• Working solution preparation:

  • Dilute in fresh buffer immediately before use

  • Do not store diluted antibody for extended periods

Following these guidelines will help maintain antibody performance and ensure reproducible results across experiments .

How should I select controls for BCL2L14 antibody experiments in different experimental systems?

Appropriate controls are essential for reliable BCL2L14 antibody experiments:

• Positive tissue/cell controls:

  • Human: Testis, lung tissue, prostate cancer tissue, A549 cells, MCF-7 cells, Jurkat cells

  • Mouse: Small intestine, colon, kidney tubules

  • Rat: Colon glandular epithelium

• Negative tissue/cell controls:

  • Mouse: Brain, liver, kidney (reported to lack BCL2L14 expression)

  • Primary cells from tissues with no expression can serve as biological negative controls

• Technical controls:

  • Primary antibody omission: Replace primary antibody with antibody diluent

  • Isotype control: Use non-specific antibody of the same isotype (e.g., Rabbit IgG monoclonal [EPR25A])

  • Loading controls for Western blot: Use housekeeping proteins appropriate for the tissue/cell type

• Validation controls:

  • Knockdown/knockout: Cells with BCL2L14 expression reduced via siRNA or CRISPR

  • Overexpression: Cells transfected with BCL2L14 expression constructs

Selecting appropriate controls based on the experimental system and application will enhance data reliability and facilitate accurate interpretation of results .

What data analysis approaches are recommended for quantitative assessment of BCL2L14 expression across different experimental platforms?

For quantitative assessment of BCL2L14 expression, researchers should consider these analysis approaches:

• Western blot quantification:

  • Densitometric analysis with normalization to housekeeping proteins

  • Multi-point standard curves using recombinant BCL2L14 protein for absolute quantification

  • Statistical comparison across multiple biological replicates (n≥3)

• Immunohistochemistry scoring:

  • H-score method (intensity × percentage of positive cells)

  • Automated image analysis with positive pixel counting algorithms

  • Blinded assessment by multiple observers

• Flow cytometry analysis:

  • Mean/median fluorescence intensity measurements

  • Comparison to isotype controls

  • Population gating strategies to identify positive/negative cells

• Immunofluorescence quantification:

  • Integrated density measurements

  • Colocalization analysis with subcellular markers

  • Z-stack analysis for 3D distribution patterns

• Cross-platform data integration:

  • Correlation analysis between protein (antibody-based) and mRNA expression data

  • Multi-omics approaches incorporating proteomics, transcriptomics, and functional data