Cleaved-BAD (D71) Antibody

What is the Cleaved-BAD (D71) Antibody and what epitope does it recognize?

Cleaved-BAD (D71) Antibody is a specialized immunological reagent designed to detect endogenous levels of fragmented Bad protein that results specifically from cleavage adjacent to the aspartic acid residue at position 71 (D71). This antibody recognizes the activated form of BAD protein that has undergone proteolytic processing at this specific site. The antibody is generated using a synthesized peptide derived from mouse BAD, typically spanning amino acids 21-70, which serves as the immunogen . The specificity of this antibody is crucial for distinguishing the cleaved, activated form from the full-length BAD protein, allowing researchers to monitor BAD activation in apoptotic pathways with precision. Both monoclonal and polyclonal versions are available, with monoclonal offering higher specificity for the cleaved form .

What is the biological significance of BAD cleavage at D71?

BAD (Bcl2 antagonist of cell death) is a critical pro-apoptotic member of the BCL-2 family that regulates programmed cell death. The cleavage of BAD at position D71 represents a significant post-translational modification that affects its function in the apoptotic cascade. When BAD undergoes cleavage at D71, it generates an activated fragment that more effectively forms heterodimers with the anti-apoptotic proteins BCL-xL and BCL-2, thereby neutralizing their death repressor activity . This activation mechanism is distinct from the well-characterized phosphorylation regulatory pathway involving protein kinases AKT and MAP kinase, as well as protein phosphatase calcineurin . The D71 cleavage event appears to be a direct activation mechanism that enhances BAD's pro-apoptotic function, particularly during apoptotic stimuli like treatment with drugs such as etoposide, as demonstrated in experimental models using 293 cells . Understanding this specific cleavage event provides insight into alternative regulatory mechanisms of the intrinsic apoptotic pathway.

How does cleaved BAD function differ from phosphorylated BAD in apoptotic pathways?

The functional differences between cleaved BAD and phosphorylated BAD represent distinct regulatory mechanisms in apoptotic signaling:

While phosphorylation of BAD (particularly at residues S112, S136, and S155) typically inactivates its pro-apoptotic function by promoting binding to cytosolic 14-3-3 proteins, cleavage at D71 appears to enhance BAD's death-promoting activity by generating a fragment that more effectively antagonizes anti-apoptotic BCL-2 family members . Experimental evidence shows that cleaved BAD can be detected in cells treated with apoptotic stimuli like etoposide, suggesting this modification occurs during active apoptotic signaling . This fundamental difference highlights the complex post-translational regulation of BAD and provides researchers with multiple markers to track apoptotic signaling in experimental systems.

What are the optimal protocols for using Cleaved-BAD (D71) Antibody in Western blot applications?

For optimal Western blot results with Cleaved-BAD (D71) Antibody, the following detailed protocol is recommended based on validated experimental parameters:

• Sample preparation:

  • Treat cells with apoptosis inducers (e.g., etoposide 25μM for 60 minutes) to generate cleaved BAD

  • Lyse cells in buffer containing protease inhibitors to prevent further degradation

  • Determine protein concentration using Bradford or BCA assay

• Gel electrophoresis and transfer:

  • Load 20-40 μg of protein per lane on 12-15% SDS-PAGE gels (higher percentage recommended for detecting the ~20kDa cleaved fragment)

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1-1.5 hours

• Blocking and antibody incubation:

  • Block membranes in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Dilute Cleaved-BAD (D71) Antibody in blocking buffer at 1:500-1:2000

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody at 1:2000-1:5000 for 1 hour at room temperature

  • Wash 3-5 times with TBST, 5 minutes each

• Detection and analysis:

  • Apply ECL substrate and detect signal using imaging system

  • Expect to observe a specific band at approximately 20kDa representing the cleaved BAD fragment

  • Always include a positive control (e.g., lysate from etoposide-treated 293 cells)

For validation of antibody specificity, perform a peptide competition assay by pre-incubating the antibody with the immunizing peptide, which should block the specific signal as demonstrated in published Western blot analyses . This rigorous approach ensures that the detected band genuinely represents cleaved BAD rather than non-specific binding or artifacts.

How can Cleaved-BAD (D71) Antibody be effectively used in immunohistochemistry (IHC) applications?

For successful immunohistochemistry applications with Cleaved-BAD (D71) Antibody, researchers should follow this optimized protocol based on experimental evidence:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process for paraffin embedding using standard protocols

  • Section at 4-6 μm thickness onto positively charged slides

  • Include positive control tissues (e.g., breast carcinoma tissue has shown detectable cleaved BAD signals)

• Antigen retrieval and blocking:

  • Deparaffinize sections in xylene and rehydrate through graded alcohols

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Allow slides to cool to room temperature for 20 minutes

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal serum in PBS for 1 hour

• Antibody incubation:

  • Dilute Cleaved-BAD (D71) Antibody at 1:100-1:300 in antibody diluent

  • Incubate sections overnight at 4°C in a humidified chamber

  • Wash 3 times with PBS, 5 minutes each

  • Apply appropriate HRP-conjugated secondary antibody system for 30-60 minutes

  • Wash 3 times with PBS, 5 minutes each

• Signal development and counterstaining:

  • Develop with DAB substrate until optimal signal-to-noise ratio is achieved (typically 2-10 minutes)

  • Counterstain with hematoxylin

  • Dehydrate through graded alcohols, clear in xylene, and mount with permanent mounting medium

• Validation controls:

  • Include a peptide competition control by pre-incubating the antibody with the immunizing peptide

  • Compare normal and apoptotic tissues to confirm specificity of staining

The cleaved-BAD signal typically appears as brown staining in cells undergoing apoptosis, with particular enrichment in tissues with high rates of programmed cell death. The distribution pattern may be cytoplasmic or mitochondrial, reflecting the localization of activated BAD during apoptotic signaling.

What are the recommended procedures for using Cleaved-BAD (D71) Antibody in cell-based ELISA assays?

Cell-based ELISA provides a high-throughput quantitative method for measuring cleaved BAD levels directly in cultured cells. Based on the available cell-based colorimetric ELISA kit protocols, the following procedure is recommended:

• Cell preparation and treatment:

  • Seed cells in 96-well clear-bottom microplates at 1-5 × 10^4 cells per well

  • Allow cells to adhere for 24-48 hours

  • Treat cells with appropriate apoptotic stimuli (e.g., etoposide, staurosporine) in experimental groups

  • Include untreated control groups for baseline comparison

• Fixation and blocking:

  • Fix cells with 4% paraformaldehyde for 20 minutes at room temperature

  • Wash 3 times with PBS

  • Permeabilize cells with 0.1% Triton X-100 in PBS for 10 minutes

  • Wash 3 times with PBS

  • Block with provided blocking buffer for 1-2 hours at room temperature

• Antibody incubation:

  • Dilute Cleaved-BAD (D71) primary antibody at 1:100 in antibody diluent

  • Add 100 μl diluted antibody to each well

  • Incubate overnight at 4°C

  • Wash 4 times with washing buffer

  • Add 100 μl of HRP-conjugated secondary antibody

  • Incubate for 1-2 hours at room temperature

  • Wash 4 times with washing buffer

• Signal development and normalization:

  • Add 100 μl of TMB substrate to each well

  • Incubate for 15-30 minutes at room temperature

  • Add 100 μl of stop solution to terminate the reaction

  • Measure absorbance at 450 nm using a microplate reader

  • Perform cell normalization using Crystal Violet staining

  • Re-read the plate at 595 nm for cell number normalization

  • Calculate the relative expression level by normalizing the target protein OD values to cell counts

This cell-based ELISA approach offers several advantages over traditional Western blot, including higher throughput, quantitative results, and the ability to normalize for cell number variations between wells. For optimal results, titrate antibody concentrations and incubation times based on your specific cell types and experimental conditions.

What are common issues in detecting cleaved BAD and their solutions?

Researchers working with Cleaved-BAD (D71) Antibody may encounter several technical challenges. The following table outlines common problems and their methodological solutions:

When troubleshooting, always include both positive controls (e.g., etoposide-treated cells known to express cleaved BAD) and negative controls (primary antibody omitted or pre-absorbed with immunizing peptide) to validate that observed signals are specific to cleaved BAD.

How can researchers validate the specificity of Cleaved-BAD (D71) Antibody in experimental systems?

Validating antibody specificity is crucial for ensuring reliable experimental results. For Cleaved-BAD (D71) Antibody, the following comprehensive validation approach is recommended:

• Peptide competition assay:

  • Pre-incubate the Cleaved-BAD (D71) Antibody with the synthesized immunizing peptide (from mouse BAD, AA range 21-70)

  • Apply the peptide-blocked antibody in parallel with the unblocked antibody

  • The specific signal should be significantly reduced or eliminated in the peptide-blocked sample

  • This has been demonstrated in both Western blot and IHC applications as shown in published results

• Genetic validation:

  • Compare signal between wild-type cells and BAD-knockout cells

  • Alternatively, use siRNA or shRNA to knockdown BAD expression

  • The specific cleaved-BAD signal should be absent or significantly reduced in knockout/knockdown samples

  • Re-expression of wild-type BAD should restore the signal

• Apoptotic stimuli comparison:

  • Compare untreated cells with cells treated with known apoptotic stimuli (e.g., etoposide 25μM for 60 minutes)

  • Include time-course experiments to track the appearance of cleaved BAD

  • Use pan-caspase inhibitors (e.g., Z-VAD-FMK) to confirm that cleavage is caspase-dependent

  • Correlate cleaved-BAD detection with other apoptotic markers (e.g., cleaved PARP, activated caspases)

• Parallel antibody validation:

  • Compare results using different antibody clones targeting cleaved BAD

  • Use both monoclonal (e.g., YP-mAb-00023) and polyclonal (e.g., CSB-PA000035) antibodies

  • Consistent results across different antibodies strengthen confidence in specificity

• Multiple detection methods:

  • Validate findings across multiple techniques (Western blot, IHC, IF, ELISA)

  • Each method should show consistent patterns of cleaved BAD detection

  • Co-localization studies in IF can confirm expected subcellular distribution during apoptosis

Thorough validation ensures that experimental observations reflect the true biological activity of cleaved BAD rather than artifacts or non-specific binding, leading to more reliable and reproducible research outcomes.

What are the critical considerations when interpreting cleaved BAD expression data?

When analyzing data generated using Cleaved-BAD (D71) Antibody, researchers should consider several critical factors that influence accurate interpretation:

By carefully considering these factors, researchers can avoid common pitfalls in data interpretation and extract meaningful biological insights from experiments using Cleaved-BAD (D71) Antibody. This contextual understanding is particularly important when studying the complex regulatory networks governing apoptotic signaling.

How can Cleaved-BAD (D71) Antibody be used to study the relationship between apoptosis and other cellular processes?

Cleaved-BAD (D71) Antibody offers sophisticated applications for investigating the interplay between apoptotic signaling and other cellular processes:

• Autophagy-apoptosis crosstalk:

  • Use dual immunostaining with cleaved BAD and autophagy markers (LC3, p62)

  • Determine whether autophagy induction affects BAD cleavage patterns

  • Investigate whether pharmacological modulators of autophagy (rapamycin, chloroquine) alter BAD processing

  • Compare cleaved BAD levels in autophagy-deficient (ATG5/7 knockout) and wild-type cells

• Cell cycle regulation:

  • Combine cleaved BAD detection with cell cycle analysis (flow cytometry, EdU incorporation)

  • Synchronize cells at different cell cycle phases and measure BAD cleavage susceptibility

  • Determine whether cell cycle checkpoint inhibitors affect BAD processing

  • Correlate CDK activity with BAD cleavage to identify potential regulatory mechanisms

• Metabolic stress responses:

  • Examine BAD cleavage under various metabolic challenges (glucose deprivation, hypoxia)

  • Investigate the connection between energy stress (AMPK activation) and BAD processing

  • Determine whether BAD cleavage affects mitochondrial metabolism using Seahorse analysis

  • Study how lipid metabolism influences BAD cleavage, given BAD's role in lipid metabolism

• Inflammation-apoptosis interface:

  • Examine BAD cleavage patterns in response to inflammatory cytokines

  • Compare inflammatory cell death (pyroptosis, necroptosis) with apoptotic BAD processing

  • Investigate whether NF-κB activation status affects BAD cleavage

  • Use cleaved BAD as a marker to distinguish apoptotic from non-apoptotic cell death in inflammatory contexts

These advanced applications extend beyond simple detection of apoptosis to explore the complex regulatory networks connecting programmed cell death with other essential cellular processes. When designing such experiments, researchers should incorporate appropriate controls and combine cleaved BAD detection with complementary markers specific to each cellular process being investigated.

What are emerging techniques for studying cleaved BAD in complex biological systems?

Recent methodological advances have expanded the toolkit for studying cleaved BAD in sophisticated experimental systems:

• Live-cell imaging approaches:

  • FRET-based BAD cleavage reporters that change fluorescence properties upon D71 cleavage

  • Coupling with apoptotic executioner proteases (caspase reporters) to correlate timing of events

  • Integration with mitochondrial membrane potential dyes to link BAD cleavage with MOMP

  • These approaches enable real-time, single-cell tracking of BAD processing dynamics

• Single-cell analysis:

  • Single-cell Western blot techniques to examine cleaved BAD heterogeneity within populations

  • Mass cytometry (CyTOF) incorporating cleaved BAD detection for multi-parameter single-cell profiling

  • Combining with single-cell RNA-seq to correlate transcriptional states with BAD cleavage susceptibility

  • These methods reveal population heterogeneity masked in bulk analyses

• Advanced tissue analysis:

  • Multiplex immunofluorescence to simultaneously detect cleaved BAD with multiple markers

  • Spatial transcriptomics to correlate cleaved BAD with local gene expression patterns

  • Digital spatial profiling for quantitative analysis of cleaved BAD in tissue microenvironments

  • These techniques preserve spatial context critical for understanding in vivo relevance

• Proteomics integration:

  • Targeted proteomics (PRM/MRM) for absolute quantification of cleaved BAD fragments

  • Proximity labeling approaches to identify proteins interacting specifically with cleaved BAD

  • Phospho-proteomics to map the interplay between BAD cleavage and phosphorylation

  • Global proteome analysis to identify downstream effectors of cleaved BAD signaling

• Organoid and 3D culture systems:

  • Detection of cleaved BAD in patient-derived organoids for personalized medicine applications

  • Spatial mapping of apoptotic gradients in 3D tumor spheroids using cleaved BAD staining

  • Microfluidics-based organ-on-chip systems to study BAD cleavage under physiological flow conditions

  • These models bridge the gap between traditional cell culture and animal models

These emerging technologies enable researchers to move beyond conventional detection methods to gain deeper insights into the spatiotemporal dynamics and functional consequences of BAD cleavage in complex biological systems. When implementing these advanced approaches, researchers should standardize protocols and include appropriate validation controls specific to each technique.

How does the amino acid substitution at position D71 affect BAD function and antibody recognition?

The D71 residue in BAD protein represents a critical functional site where molecular alterations significantly impact both protein function and antibody recognition:

• Structural and functional significance:

  • D71 contains a negatively charged aspartic acid side chain that influences protein conformation

  • Substitution of D71 with alanine (D71A) or asparagine (D71N) eliminates the negative charge, affecting protein-protein interactions

  • These substitutions increase the UUG:AUG ratio approximately twofold in translation initiation studies

  • The data suggests that eliminating the negative charge at this Loop 2 residue is sufficient to decrease initiation accuracy

• Impact on antibody recognition:

  • Cleaved-BAD (D71) Antibody specifically recognizes the neo-epitope created by proteolytic cleavage adjacent to D71

  • Amino acid substitutions at D71 would likely prevent antibody recognition, creating a useful negative control

  • The cleaved fragment detected by this antibody (approximately 20kDa) represents the portion of BAD after the D71 cleavage site

  • Researchers can leverage this specificity to distinguish between wild-type and mutant forms in experimental systems

• Experimental implications:

  • Site-directed mutagenesis of D71 creates BAD variants resistant to this specific cleavage event

  • D71A or D71N mutants can be used to study the specific role of D71 cleavage versus other regulatory mechanisms

  • Comparing cells expressing wild-type BAD versus D71-mutant BAD provides insights into cleavage-specific functions

  • Differential sensitivity to apoptotic stimuli between wild-type and D71-mutant expressing cells reveals the contribution of this specific cleavage to cell death pathways

• Advanced structure-function analyses:

  • Molecular dynamics simulations can predict how D71 substitutions alter BAD's interaction with binding partners

  • Experiments comparing negatively charged (D), neutral polar (N), and hydrophobic (A) substitutions reveal the physicochemical basis of D71's function

  • Crystal structure studies with BCL-2 family proteins can visualize how D71 region cleavage alters binding interfaces

  • Positively charged substitutions (D71R/K) would likely have more dramatic effects than charge elimination alone

This detailed understanding of the D71 position enables researchers to design sophisticated experiments that distinguish between different modes of BAD regulation and create targeted mutations that specifically affect cleavage-dependent functions while preserving other aspects of BAD biology.

How does Cleaved-BAD (D71) detection compare with other apoptotic markers in various experimental systems?

Selecting appropriate apoptotic markers requires understanding their relative advantages in different experimental contexts:

For optimal experimental design, researchers should consider using Cleaved-BAD (D71) Antibody in combination with complementary markers. For example:

• Mechanistic studies of intrinsic apoptosis: Combine cleaved BAD (early event) with cytochrome c release (mid event) and cleaved caspase-3 (late event) to track the complete pathway

• Drug sensitivity screening: Use cleaved BAD to identify compounds that specifically target BCL-2 family-regulated apoptosis versus other death mechanisms

• Tissue analysis: Multiplex cleaved BAD with TUNEL and cleaved caspase-3 to distinguish between cells at different apoptotic stages within the same tissue section

This comparative approach provides a more comprehensive picture of apoptotic signaling than any single marker alone and helps researchers select the most appropriate detection methods for their specific experimental questions.

What are the best experimental designs for studying the relationship between BAD cleavage and phosphorylation?

To effectively investigate the complex interplay between BAD cleavage and phosphorylation, researchers should consider these strategic experimental approaches:

• Sequential post-translational modification analysis:

  • Design time-course experiments tracking both BAD phosphorylation (at S112, S136, S155) and D71 cleavage

  • Use phospho-specific BAD antibodies alongside Cleaved-BAD (D71) Antibody

  • Determine whether phosphorylation precedes or follows cleavage events

  • Investigate whether one modification affects susceptibility to the other

• Mutation-based dissection:

  • Generate BAD variants with mutations at key sites:

    • Phosphorylation-deficient mutants (S112A, S136A, S155A)

    • Cleavage-resistant mutant (D71A or D71N)

    • Combined mutants affecting both modifications

  • Compare apoptotic responses, protein interactions, and subcellular localization across mutants

  • Determine whether phosphorylation status affects cleavage efficiency and vice versa

• Kinase/phosphatase manipulation:

  • Modulate key BAD-regulatory enzymes:

    • AKT inhibitors/activators to alter S136 phosphorylation

    • PKA modulators to change S112 phosphorylation

    • Calcineurin inhibitors (cyclosporin A, FK506) to prevent dephosphorylation

  • Monitor resulting effects on BAD cleavage patterns

  • Perform reciprocal experiments with caspase inhibitors to determine effects on phosphorylation

• Spatial regulation studies:

  • Investigate subcellular compartmentalization using fractionation approaches

  • Determine whether cleaved BAD shows different phosphorylation patterns than intact BAD

  • Use immunofluorescence microscopy to visualize co-localization of cleaved and phosphorylated forms

  • Develop FRET-based biosensors to monitor both modifications simultaneously in living cells

• Binding partner analysis:

  • Compare interaction profiles of cleaved versus phosphorylated BAD using co-immunoprecipitation

  • Examine competition between 14-3-3 proteins (bind phospho-BAD) and BCL-2/BCL-xL (bind cleaved BAD)

  • Perform in vitro binding assays with recombinant proteins to determine binding affinities

  • Use proximity ligation assays to visualize these interactions in situ