A6 Antibody

What are the different types of A6 antibodies used in research?

Multiple antibodies share the "A6" designation, each targeting distinct molecular entities. These include:

• Annexin A6 antibodies that recognize endogenous levels of total annexin A6 protein with reactivity across human, mouse, and rat species

• Monoclonal antibody A6 recognizing specific CD45RO epitopes on T-cells

• Clone A6 antibodies targeting NOTCH1, with cross-reactivity between human and mouse species

• Antibodies binding to conformational epitopes on the extracellular interferon gamma receptor (IFNγR)

Understanding which A6 antibody you are working with is critical for experimental design and interpretation of results, as these antibodies have distinct applications and recognition profiles.

How do A6 antibodies against Annexin A6 differ from other annexin antibodies?

Annexin A6 antibodies specifically recognize the 72 kDa Annexin A6 protein that mediates Ca²⁺-dependent binding to phospholipids . Unlike antibodies targeting other annexin family members, A6 antibodies against Annexin A6 recognize a protein predominantly located at the plasma membrane and endosomal compartments where it regulates cell migration and endosome trafficking .

These antibodies have been validated for detection in various systems:

• Western blot detection in multiple cell lines including Jurkat (human), NIH-3T3 (mouse), and NRK (rat)

• Immunohistochemistry in human placenta, specifically localizing to membranes of syncytiotrophoblast cells

• Detection in extracellular vesicles from tumor microenvironments

This specificity makes them valuable tools for investigating membrane domain organization, signaling complex interactions, and cholesterol homeostasis mechanisms.

What epitopes does the A6 monoclonal antibody targeting CD45RO recognize?

The A6 monoclonal antibody recognizes a unique epitope strongly expressed on the lower molecular weight isoform (p180) of Leukocyte Common Antigen (LCA/CD45), but also weakly expressed on the p190 isoform coded by exon B and the p205 coded by exons A and B . This epitope is:

• Carbohydrate-dependent (neuraminidase-sensitive)

• Trypsin-resistant

• Fixation and paraffin-embedding-resistant

What are the optimal conditions for using A6 antibodies in Western blotting?

For Western blot applications, the following conditions have been reported as optimal based on published research:

For Annexin A6 Antibodies:

• Dilution: 1:1000 recommended for polyclonal Annexin A6 antibodies

• For monoclonal antibodies (e.g., MAB5186), 0.1 μg/mL concentration has been validated

• Buffer system: Immunoblot Buffer Group 2 has been successfully used

• Detection system: HRP-conjugated secondary antibodies show good results

• Expected band size: ~70-72 kDa under reducing conditions

For Simple Western™ automated capillary-based system:

• Loading concentration: 0.5 mg/ml of lysate

• Antibody concentration: 1 μg/mL

• Separation system: 12-230kDa system works effectively

Validation across multiple cell lines is recommended to ensure specificity, as demonstrated in tests with Jurkat, NIH-3T3, and NRK cell lines .

How should A6 antibodies be used for immunohistochemistry (IHC) applications?

For IHC applications with A6 antibodies, the following protocol has been validated:

For Annexin A6 detection in paraffin-embedded tissues:

• Fixation: The antibody works with immersion-fixed paraffin-embedded sections

• Antigen retrieval: Heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic is recommended

• Antibody concentration: 15 μg/mL

• Incubation conditions: Overnight at 4°C

• Detection system: HRP-DAB staining kit, with hematoxylin counterstaining

For CD45RO detection with A6 monoclonal antibody:

• The A6 antibody maintains reactivity across multiple fixation methods including formalin, Bouin's fluid, Carnoy's fixative, and B5

• Resistant to decalcification procedures

• Trypsin digestion slightly enhances A6 reactivity

• Expected staining pattern: Most T lymphocytes, macrophages, and Langerhans' cells in normal tissues

The preservation of A6 reactivity across different fixatives makes it particularly valuable for retrospective studies on archived pathological specimens.

How do I determine the specificity of A6 antibodies in my experimental system?

Validating antibody specificity is crucial for accurate interpretation of results. For A6 antibodies, consider these approaches:

• Cross-species reactivity testing:

  • Test in human, mouse, and rat systems if working with Annexin A6 antibodies

  • Document expected molecular weight differences between species

• Multiple detection techniques:

  • Compare results across Western blot, IHC, and flow cytometry

  • For Annexin A6, validate 72 kDa band in Western blot corresponds to staining patterns in IHC

• Cell type specificity:

  • For A6 CD45RO antibodies, confirm appropriate staining of T lymphocytes but not B cells or NK cells

  • For Annexin A6, verify membrane and endosomal localization

• Enzyme treatment controls:

  • Test sensitivity to neuraminidase for CD45RO A6 antibodies (should reduce signal)

  • Test resistance to trypsin (signal should be maintained)

• Cross-blocking experiments:

  • For CD45RO detection, compare with established antibodies like UCHL-1

  • Document differences in cell population labeling efficiency

How can A6 antibodies be used to study extracellular vesicles in cancer research?

Annexin A6 is frequently upregulated in extracellular vesicles (EVs) within the tumor microenvironment, particularly after chemotherapy or tyrosine kinase inhibitor treatment . A6 antibodies provide valuable tools for investigating these processes:

Research applications include:

• Monitoring therapy-induced changes in EV composition:

  • Track Annexin A6 levels in EVs before and after treatment

  • Correlate with resistance development and metastatic potential

• Investigating resistance mechanisms:

  • Annexin A6 in EVs enhances stability of membrane-associated receptor tyrosine kinases (RTKs), such as EGFR

  • A6 antibodies can be used to immunoprecipitate and study these complexes

• Cancer cell phenotype studies:

  • Investigate how Annexin A6-rich EVs influence:

    • Cell motility

    • Stemness

    • Autophagy induction

• Tumor microenvironment communication:

  • Track EV transfer between cells using Annexin A6 as a marker

  • Study in multiple cancer types including lung, breast, and pancreatic duct adenocarcinoma

This approach has revealed that Annexin A6-enriched EVs promote resistance and metastasis across multiple cancer types, making A6 antibodies crucial tools for translational cancer research.

What are the key considerations when using A6 antibodies to study T cell memory and activation?

The A6 monoclonal antibody recognizing CD45RO isoforms has specific applications in T cell research:

• Identification of functional T cell subsets:

  • A6 stains TCR-αβ+ cells with differential intensities, subdividing them into bright and dim populations

  • Strongly stains all TCR-γδ+ cells

  • Does not stain CD19+ B cells or CD56+ NK cells

• Functional studies:

  • Depletion experiments: A6 depletion dramatically decreases proliferative responses to recall antigens and anti-CD3 mAb

  • Unlike UCHL1 (another CD45RO antibody), A6 also depletes alloreactive T cells affecting primary and secondary mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML)

• Methodological considerations:

  • Differential staining intensity must be accurately gated in flow cytometry

  • Include appropriate isotype controls

  • Consider dual staining with other T cell markers to define subpopulations

• Applications in pathology:

  • Effective for identifying T cell lymphomas (31 of 34 peripheral T-cell lymphomas tested positive)

  • Useful for Ki-1+ lymphomas (12 of 18 positive)

  • Limited utility in T-cell leukemias (only 1 of 5 positive)

The ability of A6 to recognize memory, activated, and alloreactive T cells makes it particularly valuable for immunological research and diagnostic applications.

How do epitope binding characteristics affect experimental design when using the A6 antibody targeting the IFNγ receptor?

The A6 antibody that binds to the extracellular interferon gamma receptor (IFNγR) has unique epitope binding characteristics that influence experimental design:

• Conformational epitope considerations:

  • A6 binds a conformational epitope primarily comprising the CC' surface loop on the N-terminal fibronectin type-III domain of IFNγR

  • Non-denaturing conditions must be maintained for experiments requiring epitope recognition

• Interface characteristics:

  • The crystal structure reveals an interface rich in aromatic side chains (Trp, Tyr, His)

  • Energetically important side chains (ΔG > 2.4 kcal/mol) form distinct "hot spots" in the binding interface

  • Key interactions include:

    • VLW92 in A6 interacts with K47 in receptor via π-cation interaction

    • VLW92 simultaneously T-stacks onto W82 indole ring in receptor

    • VHW52, VHW53, VHD54, VHD56 surround aliphatic side chains

• Experimental implications:

  • Mutations in hot spot residues dramatically reduce binding affinity

  • Buffer conditions must maintain proper protein folding

  • Surface plasmon resonance is an effective method to measure binding kinetics

  • When designing blocking experiments, consider that A6 binding may not directly compete with IFNγ binding

Understanding these epitope characteristics is essential when using A6 antibodies in structure-function studies, binding assays, or when engineering antibody derivatives with modified binding properties.

What strategies can resolve inconsistent Western blot results with A6 antibodies?

Inconsistent Western blot results with A6 antibodies may be addressed through several optimization strategies:

• Protein extraction and sample preparation:

  • For Annexin A6 detection, use a buffer system that preserves protein conformation

  • Maintain reducing conditions for consistent 70-72 kDa band detection

  • Ensure complete solubilization of membrane-associated proteins

• Antibody concentration optimization:

  • For monoclonal A6 antibodies (MAB5186), test concentration range around 0.1 μg/mL

  • For polyclonal antibodies, optimize around 1:1000 dilution

  • Consider extended incubation times (overnight at 4°C) for weak signals

• Buffer system adjustments:

  • For Annexin A6 detection, Immunoblot Buffer Group 2 has been validated

  • Test alternative blocking agents if background is high

• Detection system considerations:

  • HRP-conjugated secondary antibodies are generally effective

  • For low abundance targets, consider enhanced chemiluminescence systems

  • For multiple band issues, validate with knockout/knockdown controls

• Cross-validation approaches:

  • Compare results with alternative antibodies against the same target

  • Verify expected molecular weight (70-72 kDa for Annexin A6)

  • Consider alternative detection methods like Simple Western™ automated systems

How can immunohistochemical staining with A6 antibodies be optimized for difficult tissue samples?

Optimizing immunohistochemical staining with A6 antibodies for challenging tissue samples:

• Fixation and antigen retrieval optimization:

  • For CD45RO A6 antibodies: These are remarkably resistant to different fixatives (formalin, Bouin's fluid, Carnoy's fixative, B5)

  • For Annexin A6: Heat-induced epitope retrieval using basic retrieval reagents is recommended

  • Consider extended antigen retrieval times for heavily fixed samples

• Enzymatic enhancement:

  • For CD45RO A6 antibodies: Trypsin digestion slightly enhances reactivity

  • Test brief neuraminidase treatment for other A6 antibodies to potentially expose hidden epitopes

• Antibody concentration and incubation:

  • For Annexin A6: 15 μg/mL with overnight incubation at 4°C is effective

  • For difficult samples, consider:

    • Extended incubation times (up to 48 hours at 4°C)

    • Step-wise increasing antibody concentration

• Signal amplification:

  • Consider tyramide signal amplification systems for weak signals

  • Explore polymer-based detection systems for improved sensitivity

  • Balance amplification with background signal optimization

• Background reduction:

  • Include additional blocking steps (avidin-biotin blocking if using biotinylated reagents)

  • Pre-absorb secondary antibodies if non-specific binding is observed

  • Include appropriate controls on serial sections

The A6 antibody against CD45RO has shown particular value for archived and difficult pathological specimens due to its resistance to various fixation methods .

What controls are critical when using A6 antibodies in multiparameter flow cytometry?

When using A6 antibodies in multiparameter flow cytometry, these controls are essential:

• Isotype controls:

  • Match isotype, concentration, and fluorochrome to the A6 antibody

  • Particularly important when analyzing differential staining intensity seen with CD45RO A6 antibodies

• Fluorescence minus one (FMO) controls:

  • Critical for setting accurate gates when using A6 antibodies that show differential staining intensities

  • Essential when the A6 antibody is part of a large multicolor panel

• Known positive and negative populations:

  • For CD45RO A6 antibodies:

    • Positive controls: T lymphocytes, macrophages

    • Negative controls: CD19+ B cells, CD56+ NK cells

  • For Annexin A6: Test in cell lines with known expression (e.g., Jurkat cells)

• Enzyme treatment controls:

  • Neuraminidase treatment should reduce signal for CD45RO A6 antibodies (epitope is carbohydrate-dependent)

  • Trypsin treatment should maintain signal (epitope is trypsin-resistant)

• Compensation controls:

  • Single-stained controls for each fluorochrome

  • Include compensation beads alongside cellular controls

  • Verify compensation settings do not distort differential staining patterns

• Viability discrimination:

  • Include viability dye to exclude dead cells that may bind antibodies non-specifically

  • Particularly important when studying activated T cell populations that may have increased apoptosis

These controls enable accurate interpretation of staining patterns, particularly for A6 antibodies that show differential intensity on functional T cell subpopulations .

How are A6 antibodies being used to study extracellular vesicle-mediated therapy resistance in cancer?

Recent research has revealed important roles for Annexin A6 in therapy resistance mechanisms, leading to novel applications of A6 antibodies:

• Monitoring therapy-induced EV changes:

  • A6 antibodies detect increased Annexin A6 in EVs following chemotherapy or tyrosine kinase inhibitor treatment

  • This allows tracking of therapy-induced adaptations in real-time

• Mechanistic studies of resistance:

  • Annexin A6 in EVs stabilizes membrane-associated receptor tyrosine kinases (RTKs) like EGFR

  • A6 antibodies enable visualization and quantification of these complexes via immunoprecipitation and immunofluorescence

• Functional characterization of EVs:

  • A6 antibodies help stratify EV populations based on Annexin A6 content

  • This facilitates studies correlating Annexin A6-rich EVs with:

    • Enhanced cancer cell motility

    • Stemness properties

    • Autophagy induction

• Targeting approaches:

  • A6 antibodies are being explored to neutralize Annexin A6-rich EVs

  • This may potentially overcome therapy resistance mechanisms

In lung cancer, breast cancer, and pancreatic duct adenocarcinoma, these approaches have revealed that Annexin A6-enriched EVs promote resistance and metastasis following therapy , opening new avenues for therapeutic intervention.

What insights have structural studies of the A6 antibody-antigen interface provided for antibody engineering?

Structural analysis of the A6 antibody binding to the extracellular interferon gamma receptor has revealed important principles for antibody engineering:

• Hot spot identification:

  • Alanine-scanning mutagenesis identified key residues where ΔG(mutant) - ΔG(wt) > 2.4 kcal/mol

  • These energetically important residues form distinct "hot spots" at the binding interface

  • Knowledge of these hot spots guides rational antibody optimization

• Role of aromatic interactions:

  • The interface is rich in aromatic side chains (Trp, Tyr, His)

  • These aromatic residues interact with both polar and hydrophobic groups

  • This versatility in interaction types appears advantageous for ligand binding

• Specific interaction motifs:

  • VLW92 in A6 participates in a π-cation interaction with K47 in the receptor

  • Simultaneously, VLW92 T-stacks onto the W82 indole ring

  • VHW52, VHW53, VHD54, and VHD56 surround aliphatic side chains

• Engineering applications:

  • These insights guide site-directed mutagenesis to enhance binding affinity

  • Enable development of antibody variants with modified specificity

  • Inform computational antibody design approaches

The combination of crystal structure analysis with alanine-scanning mutagenesis and surface plasmon resonance measurements provides a powerful approach for understanding antibody-antigen interactions at the molecular level .

How do A6 CD45RO antibodies compare with other CD45 isoform-specific antibodies in diagnostic applications?

Comparative analysis of A6 CD45RO antibodies with other CD45 isoform-specific antibodies reveals important distinctions for diagnostic applications:

The A6 antibody demonstrates several advantages for diagnostic applications:

• Superior resistance to different fixation methods

• Labels a higher percentage of cells in pathological tissues than UCHL-1

• Effectively identifies T cell lymphomas with high sensitivity

• Additionally detects alloreactive T cells not captured by UCHL-1

These characteristics make A6 an excellent reagent for detection of CD45RO in paraffin-embedded normal and pathologic tissues , particularly for challenging archived specimens.

What criteria should guide selection between monoclonal and polyclonal A6 antibodies for specific applications?

When choosing between monoclonal and polyclonal A6 antibodies, consider these application-specific criteria:

Western Blotting:

• Monoclonal advantages:

  • Higher specificity for single epitope (e.g., MAB5186 at 0.1 μg/mL)

  • Consistent lot-to-lot reproducibility

• Polyclonal advantages:

  • Potentially higher sensitivity (recognizing multiple epitopes)

  • May be more resistant to epitope loss from denaturation

  • Typical working dilution around 1:1000

Immunohistochemistry:

• Monoclonal advantages:

  • For CD45RO detection, A6 monoclonal maintains reactivity across multiple fixatives

  • Consistent staining patterns (15 μg/mL concentration validated)

• Polyclonal considerations:

  • May provide signal amplification for low-abundance targets

  • Higher potential for background staining

Immunoprecipitation:

• Monoclonal advantages:

  • Higher specificity for confirming protein interactions

  • Lower background in downstream applications

• Polyclonal advantages:

  • More efficient capture of target proteins

  • Better performance when epitopes may be partially masked

Flow Cytometry:

• Monoclonal advantages:

  • Critical for detecting differential staining intensities (as seen with A6 CD45RO antibody)

  • More predictable titration for multicolor panels

• Polyclonal considerations:

  • Generally less suitable for flow cytometry applications

Structure-Function Studies:

• Monoclonal advantages:

  • Well-defined epitope binding (as with A6 anti-IFNγR)

  • Essential for crystallography studies

  • Enables precise epitope mapping

Selection should be guided by the specific research question, target abundance, and experimental conditions, with careful validation in your specific experimental system.

How might A6 antibodies contribute to developing liquid biopsy approaches for cancer monitoring?

Emerging research on Annexin A6 in extracellular vesicles suggests promising applications for A6 antibodies in liquid biopsy development:

• EV-based biomarker detection:

  • Annexin A6 is upregulated in EVs from the tumor microenvironment after therapy

  • A6 antibodies could enable capture and quantification of these therapy-induced EVs from blood

  • Potential for early detection of resistance development

• Multiparameter EV characterization:

  • A6 antibodies combined with other markers could create EV "fingerprints"

  • This approach may distinguish EVs from different cellular origins

  • Applications in multiple cancer types (lung, breast, pancreatic)

• Functional assessment:

  • Captured Annexin A6-rich EVs could be tested for:

    • EGFR stability enhancement

    • Promotion of cell motility

    • Induction of stemness and autophagy

  • These functional readouts may have prognostic value

• Therapeutic monitoring:

  • Serial sampling could track changes in Annexin A6-EV profiles during treatment

  • Potential to guide treatment adjustments before clinical progression

  • May help identify patients requiring combination approaches

While still in early research stages, the clear upregulation of Annexin A6 in therapy-induced EVs across multiple cancer types suggests significant potential for A6 antibodies in developing non-invasive monitoring approaches.

What are the potential applications of A6 antibodies in studying conformational epitopes for vaccine development?

The unique properties of the A6 antibody binding to the interferon gamma receptor offer insights for vaccine development approaches:

• Conformational epitope mapping:

  • A6 binds a conformational epitope comprising mainly the CC' surface loop on the N-terminal fibronectin type-III domain

  • Similar approaches could map protective conformational epitopes on pathogen proteins

• Structure-guided immunogen design:

  • The crystal structure of the A6-IFNγR complex revealed interface characteristics rich in aromatic side chains

  • These insights could guide design of immunogens that present critical conformational epitopes

  • Applications in designing vaccines against structurally complex targets

• Antibody engineering approaches:

  • Alanine-scanning mutagenesis identified "hot spots" in the binding interface

  • Similar approaches could enhance vaccine-induced antibody binding to key epitopes

  • Potential for designing immunogens that elicit antibodies with specific functional properties

• Validation methodologies:

  • Surface plasmon resonance techniques used to measure A6 binding affinities

  • Similar approaches could evaluate vaccine-induced antibody qualities

  • May help identify correlates of protection for conformational epitope-targeted vaccines

These applications leverage the detailed understanding of A6 antibody binding characteristics to advance structure-based vaccine design approaches, particularly for targets where conformational epitopes are critical for protection.

How might advances in A6 antibody research inform therapeutic antibody development?

Insights from A6 antibody research provide valuable principles for therapeutic antibody development:

• Interface optimization principles:

  • The interface rich in aromatic side chains (Trp, Tyr, His) in the A6-IFNγR complex suggests design strategies

  • These aromatic residues interact with both polar and hydrophobic groups, providing versatile binding

  • Similar approaches could enhance therapeutic antibody binding to challenging targets

• Hot spot targeting:

  • Identification of energetically important residues (ΔG > 2.4 kcal/mol) that form distinct binding hot spots

  • Therapeutic antibodies could be engineered to optimize interactions at similar hot spots

  • Potential for enhanced potency and specificity

• Specific interaction motifs:

  • π-cation interactions and aromatic stacking (as seen between VLW92 in A6 and receptor residues)

  • Engineering these interaction types into therapeutic antibodies may enhance binding properties

  • Applications in targeting difficult epitopes on therapeutic targets

• Diagnostic-therapeutic combinations:

  • A6 antibodies against Annexin A6 could potentially:

    • Identify patients with Annexin A6-rich EVs associated with resistance

    • Target these EVs to overcome resistance mechanisms

    • Monitor treatment response through EV profiling

• Cross-species reactivity considerations:

  • The cross-species reactivity of some A6 antibodies (human, mouse, rat)

  • Facilitates translational research from preclinical to clinical applications

  • Important for developing antibodies with similar cross-species binding profiles