APO2 Antibody
Description
Introduction to APO2 Antibody
The APO2.7 (7A6) antibody is a monoclonal IgG1 mouse antibody that detects the APO2.7 antigen, a 38 kDa mitochondrial membrane protein expressed during apoptosis . This antibody serves as a critical tool for identifying apoptotic cells in research, particularly in cancer studies, by targeting the 7A6 antigen—a marker restricted to cells undergoing programmed cell death . Unlike other apoptosis markers, APO2.7 expression occurs early in the apoptotic process, making it valuable for detecting initial stages of cell death .
Mechanism of Action
Target Antigen: The APO2.7 antigen is localized on the inner mitochondrial membrane and becomes exposed during apoptosis . Its expression correlates with mitochondrial membrane permeability changes, a hallmark of apoptotic signaling .
Detection Workflow:
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Cells are permeabilized (e.g., with digitonin) to allow antibody access to intracellular mitochondrial antigens .
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APO2.7 binds specifically to the 7A6 antigen, enabling quantification of apoptotic cells via flow cytometry .
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Normal viable cells show minimal or no APO2.7 staining, whereas apoptotic cells exhibit strong positivity .
Table 1: APO2.7 Antibody Characteristics
Table 2: APO2.7 in Hypoxia-Induced Apoptosis (Breast Cancer Model)
Applications in Cancer Research
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Early Apoptosis Detection: APO2.7 reliably identifies apoptosis in tumor cells treated with chemotherapy, radiation, or death receptor agonists (e.g., Fas/CD95) .
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Combination Therapies: Used to assess synergy between pro-apoptotic agents (e.g., TRAIL/Apo2L) and chemotherapeutics like 5-fluorouracil .
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Tumor Microenvironment Studies: APO2.7 helps map apoptotic heterogeneity in hypoxic tumor regions, informing resistance mechanisms .
Clinical and Preclinical Relevance
While APO2.7 itself is not a therapeutic agent, it is instrumental in preclinical drug development:
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Validates efficacy of TRAIL/Apo2L-based therapies by quantifying tumor cell apoptosis .
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Monitors treatment response in xenograft models, correlating apoptosis levels with tumor regression .
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Identifies resistant cell populations in solid tumors, guiding combination therapy strategies .
Limitations and Considerations
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Specificity: APO2.7 does not stain necrotic cells, distinguishing apoptosis from necrosis .
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Technical Requirements: Requires cell permeabilization for mitochondrial antigen access, limiting use in intact tissue samples .
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Context-Dependent Expression: Sensitivity varies by cell type and apoptosis trigger, necessitating validation across models .
Product Specs
Target Background
Q&A
What is the APO2.7 antigen and how is it related to apoptosis?
The APO2.7 antigen (also named 7A6 antigen) is a 38 kDa protein localized on the membrane of mitochondria whose expression appears to be restricted to cells undergoing apoptosis. The antigen can be detected after various apoptosis induction methods including CD95/Fas ligation, irradiation, and drug treatment. Its expression is considered an early event in the apoptosis process, while normal viable cells are either negative or only weakly positive for this marker. Research indicates that less than 2% of peripheral T cells from normal donors express the APO2.7 antigen .
What are the key specifications of the APO2.7 antibody commonly used in research?
The research-grade APO2.7 antibody has the following specifications:
| Specification | Details |
|---|---|
| Clone | 2.7A6A3 (APO2.7) |
| Isotype | IgG1 Mouse |
| Target Species | Human |
| Common Formats | PE-conjugated, others available |
| Applications | Flow cytometry (primary) |
The antibody is typically used for direct quantitation of apoptotic cells by flow cytometry after permeabilization with digitonin .
How should cell permeabilization be performed when using APO2.7 antibody?
For optimal results with APO2.7 antibody, cells must be properly permeabilized using digitonin before staining. This is a critical methodological step as research demonstrates significant differences in detection sensitivity between permeabilized and unpermeabilized samples. In experimental studies with Jurkat cells, processed (permeabilized) cells showed significant increases in APO2.7 staining after just 1.5 hours following CD95-induced apoptosis, while unprocessed cells showed minimal staining increases until after 6 hours post-induction .
The recommended permeabilization protocol involves:
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Harvesting cells at the desired timepoint
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Washing with PBS
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Treating with digitonin at appropriate concentration (typically 100 μg/mL)
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Incubating for 20 minutes at room temperature
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Washing to remove excess digitonin
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Proceeding with APO2.7 antibody staining
This permeabilization step is essential for accessing the mitochondrial membrane-associated antigen and obtaining accurate early apoptosis measurements .
How does APO2.7 antibody detection compare with other apoptosis markers in a time-course experiment?
In comparative time-course experiments using Jurkat cells with anti-CD95 (7C11) induced apoptosis, the following temporal sequence of detection has been observed:
| Time Post-Induction | Observed Changes by Method |
|---|---|
| 1.5 hours | Significant APO2.7 staining in permeabilized cells |
| 3 hours | Light-scatter changes (decreased forward-scatter, increased side-scatter); Annexin V staining begins |
| 4.5-6 hours | Continued increase in APO2.7 and Annexin V staining |
| >6 hours | DNA fragmentation detectable; Trypan blue positivity; APO2.7 staining in unpermeabilized cells |
This timeline demonstrates that APO2.7 staining of permeabilized cells provides one of the earliest detectable indicators of apoptosis, preceding both phosphatidylserine externalization (Annexin V binding) and DNA fragmentation. Monitoring APO2.7 expression therefore allows precise tracking of early mitochondrial events in the apoptotic cascade .
How can researchers distinguish between specific APO2.7 staining and background autofluorescence in flow cytometry?
When interpreting APO2.7 antibody staining by flow cytometry, researchers should implement several controls to distinguish specific staining from background:
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Include unstained controls from both treated and untreated samples to establish baseline autofluorescence
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Use isotype controls (Mouse IgG1 with matching fluorochrome) to identify non-specific binding
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Include both permeabilized and unpermeabilized samples as methodological controls
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Consider dual staining with complementary apoptosis markers (e.g., Annexin V)
Research has shown that normal viable cells exhibit negative or weakly positive APO2.7 staining, with background levels typically below 2% in peripheral T cells from normal donors . Significant shifts in fluorescence intensity above this baseline, particularly in permeabilized samples, indicate specific APO2.7 antigen expression associated with apoptosis induction.
What are common sources of variability in APO2.7 antibody staining and how can they be addressed?
Several factors can introduce variability in APO2.7 antibody staining results:
| Source of Variability | Mitigation Strategy |
|---|---|
| Incomplete permeabilization | Optimize digitonin concentration and incubation time; confirm permeabilization with control antibodies targeting intracellular antigens |
| Cell fixation issues | If fixation is necessary, use mild fixatives and validate that they don't affect APO2.7 epitope recognition |
| Timing of analysis | Standardize time points after apoptosis induction; consider multiple time points to capture the dynamic process |
| Cell type differences | Establish baseline APO2.7 expression for each cell type; activated T cells may show some APO2.7 expression associated with ongoing apoptotic processes |
| Antibody concentration | Titrate antibody to determine optimal concentration for specific experimental conditions |
Methodological consistency is crucial, as research demonstrates that detection sensitivity varies significantly between processed and unprocessed samples, and timing relative to apoptosis induction is critical for accurate interpretation .
How can APO2.7 antibody be integrated with other markers for multiparametric analysis of apoptosis pathways?
For comprehensive apoptosis pathway analysis, APO2.7 antibody can be combined with other markers in multiparametric flow cytometry:
| Marker | Parameter Measured | Temporal Relationship to APO2.7 |
|---|---|---|
| APO2.7 | Mitochondrial membrane protein expression | Early event (1.5h+) with permeabilization |
| Annexin V | Phosphatidylserine externalization | Nearly parallel to APO2.7 (3h+) |
| Propidium Iodide/7-AAD | Membrane integrity loss | Later event (>6h) |
| Active Caspase-3/7 | Executioner caspase activation | Pathway-dependent timing |
| JC-1 or TMRE | Mitochondrial membrane potential | Often precedes APO2.7 expression |
This multiparametric approach enables detailed investigation of apoptotic pathway kinetics and mechanisms. Research has shown that when used with permeabilization, APO2.7 provides earlier detection than many conventional apoptosis markers, allowing for precise monitoring of the transition points in cellular commitment to apoptosis .
What considerations should be made when using APO2.7 antibody to investigate mitochondrial involvement in different apoptotic pathways?
When using APO2.7 to investigate mitochondrial involvement across different apoptotic pathways, researchers should consider:
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Pathway-specific timing: The kinetics of APO2.7 antigen expression may vary depending on whether apoptosis is induced through extrinsic (death receptor) or intrinsic (mitochondrial) pathways.
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Cell type variations: Different cell types may exhibit varying degrees of mitochondrial involvement in apoptosis. Research findings with APO2.7 in Jurkat cells (T-lymphocyte lineage) should be validated when working with other cell types.
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Mitochondrial membrane changes: APO2.7 detection should be correlated with other measures of mitochondrial function, such as cytochrome c release or changes in mitochondrial membrane potential.
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Experimental validation: For novel apoptosis inducers, establish a time-course of APO2.7 expression compared to established methods like CD95/Fas ligation, irradiation, or drug treatment as referenced in the literature .
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Permeabilization optimization: The criticality of proper permeabilization is pathway-dependent; intrinsic pathway activation may show different permeabilization requirements than extrinsic pathway activation.
These considerations enable researchers to accurately assess mitochondrial events across diverse apoptotic mechanisms, with APO2.7 serving as a valuable tool for detecting early mitochondrial protein expression changes associated with commitment to apoptosis.
What are the advantages and limitations of using APO2.7 antibody compared to newer apoptosis detection methods?
The APO2.7 antibody offers several distinct advantages in apoptosis research:
Advantages:
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Early detection capability (1.5 hours post-induction with proper permeabilization)
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Specifically targets a mitochondrial membrane protein involved in apoptosis
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Allows precise monitoring of both early and late cellular responses
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Well-validated in research with clear temporal positioning relative to other apoptotic events
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Compatible with multiparametric flow cytometry approaches
Limitations:
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Requires proper permeabilization for early detection
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Primarily validated on human samples
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Some low-level expression (less than 2%) may occur in normal peripheral T cells
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Limited to flow cytometry applications after proper sample preparation
When comparing to newer methodologies, APO2.7 remains valuable for its specific detection of a mitochondrial protein marker that appears early in the apoptotic process, providing researchers with a well-characterized tool for investigating mitochondrial involvement in programmed cell death .
What research questions about apoptosis mechanisms can be uniquely addressed using APO2.7 antibody?
The unique properties of APO2.7 antibody enable researchers to address several important questions about apoptosis mechanisms:
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Temporal mitochondrial events: What is the precise timing of mitochondrial membrane protein changes relative to other apoptotic events?
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Pathway distinction: How do mitochondrial protein expression patterns differ between extrinsic and intrinsic apoptosis pathway activation?
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Apoptosis commitment point: At what stage do cells become irreversibly committed to apoptosis, and how does this correlate with APO2.7 antigen expression?
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Therapeutic intervention windows: Can interventions that block APO2.7 antigen expression or function alter apoptotic outcomes, and what is the critical time window?
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Cell type differences: How does the expression of APO2.7 antigen during apoptosis vary across different cell types and tissues?
