GP9 Antibody

What is GP9 and why is it important in hematological research?

GP9 (Glycoprotein IX) is a small membrane glycoprotein (approximately 19 kDa) found on the surface of human platelets. It forms a 1:1 noncovalent complex with glycoprotein Ib, creating a receptor complex for von Willebrand factor. The complete receptor includes the alpha and beta subunits associated with GP9 and platelet glycoprotein V . This complex plays a crucial role in platelet activation and aggregation, making it essential for hemostasis. Studying GP9 provides valuable insights into platelet biology, thrombotic disorders, and cardiovascular conditions. Mutations in the GP9 gene are associated with Bernard-Soulier syndrome, characterized by giant platelets and bleeding tendencies, making it a significant target for hematological research .

What types of GP9 antibodies are available for research applications?

Researchers have access to several types of GP9 antibodies, each with specific characteristics suitable for different experimental applications:

• Polyclonal antibodies: These are typically produced in rabbits, such as the GP9 Rabbit Polyclonal Antibody (CAB5374), which demonstrates high reactivity with human, mouse, and rat samples . These antibodies recognize multiple epitopes on the GP9 protein.

• Monoclonal antibodies: These include mouse monoclonal antibodies like clone GR-P, which provides high specificity for human GP9 . Monoclonal antibodies offer greater consistency between experiments compared to polyclonals.

• Conjugated antibodies: Some GP9 antibodies are available with conjugations such as APC for flow cytometry applications, enabling direct detection without secondary antibodies .

The choice between these types depends on the specific research application, with each offering different advantages in terms of specificity, sensitivity, and experimental compatibility.

What are the key differences between mouse and rabbit anti-GP9 antibodies?

The source species for antibody production significantly impacts performance characteristics:

Rabbit polyclonal anti-GP9 antibodies:

• Generally demonstrate broader epitope recognition

• Often show higher affinity due to recognition of multiple epitopes

• Exhibit cross-reactivity across species (human, mouse, rat)

• Typically used in applications requiring high sensitivity like Western blot and IHC-P

• Example: CAB5374 has been validated for Western blot, IHC-P, and ELISA applications

Mouse monoclonal anti-GP9 antibodies:

• Provide higher specificity to a single epitope

• Offer excellent batch-to-batch reproducibility

• Particularly well-suited for flow cytometry applications

• Example: Clone GR-P is specifically optimized for flow cytometry and immunoprecipitation

The choice between these depends on the experimental requirements - rabbit polyclonals for broader detection and mouse monoclonals for highly specific applications with consistent results.

How should GP9 antibodies be optimized for Western blot applications?

For optimal Western blot results with GP9 antibodies, follow these methodological guidelines:

• Sample preparation: Due to GP9's membrane localization, use specialized lysis buffers containing mild detergents (such as RIPA) that effectively extract membrane proteins while preserving epitope structure.

• Reducing vs. non-reducing conditions: Since GP9 forms complexes with other proteins, comparing results under both reducing and non-reducing conditions may provide valuable information about protein interactions. Under reducing conditions, expect to observe GP9 at approximately 19 kDa .

• Dilution optimization: Start with the recommended dilution range (1:100 - 1:500 for polyclonal antibodies like CAB5374) . Perform a dilution series to determine the optimal concentration that provides specific signal with minimal background.

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBST as a starting point, but optimize if background is problematic.

• Positive controls: Include positive control samples known to express GP9, such as K-562 cells which have been validated for GP9 expression .

• Signal detection: For low-abundance GP9 detection, consider chemiluminescent substrates with extended signal duration or fluorescent secondary antibodies for quantitative analysis.

The expected molecular weight of GP9 is 19 kDa, which serves as a validation point for specific detection .

What are the optimal protocols for GP9 detection by flow cytometry?

Flow cytometry is particularly valuable for GP9 detection on platelets, requiring specific methodological considerations:

• Sample preparation: For whole blood analysis:

  • Use anticoagulated fresh blood (preferably within 2-4 hours of collection)

  • Dilute blood 1:10 in PBS containing 1% BSA to prevent platelet activation

  • Avoid vigorous mixing that could activate platelets

• Antibody concentration: For conjugated GP9 antibodies like GR-P (APC), use approximately 20 μL per 10^6 cells as a starting point . Titrate to determine optimal concentration.

• Staining protocol:

  • Include Fc receptor blocking (especially important for whole blood)

  • Stain for 20-30 minutes at room temperature in the dark

  • Wash gently to avoid platelet activation or loss

• Gating strategy:

  • Use forward/side scatter to identify platelet population

  • Consider including additional platelet markers (CD41/CD61) for co-staining

  • Analyze GP9 expression within the platelet gate

• Controls:

  • Include unstained control, isotype control (IgG2a for monoclonal GR-P), and single-stained controls if performing multi-color analysis

This protocol has been validated for human normal whole blood samples using the GP9 monoclonal antibody clone GR-P (APC) .

How can researchers effectively validate GP9 antibody specificity?

Rigorous validation of GP9 antibody specificity is crucial for reliable experimental outcomes:

• Multiple technique validation: Confirm GP9 detection across multiple techniques (Western blot, flow cytometry, IHC) where feasible, as each provides different information about specificity.

• Positive and negative controls:

  • Use cell lines with known GP9 expression (K-562 as positive control)

  • Include samples with GP9 knockdown/knockout as negative controls

  • Test tissues/cells known to lack GP9 expression

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific signal should be blocked by this competition.

• Molecular weight verification: Confirm detection at the expected 19 kDa size for GP9 .

• Expression pattern analysis: Compare observed expression patterns with published literature on GP9 localization (membrane, single-pass type I membrane protein) .

• Cross-reactivity testing: If working with multiple species, validate specificity in each species separately rather than assuming cross-reactivity.

• Antibody registry databases: Check if the antibody has been validated in community resources like the Antibody Registry or CiteAb.

This systematic validation approach ensures that experimental results reflect true GP9 biology rather than non-specific interactions.

How can GP9 antibodies be used to investigate Bernard-Soulier syndrome (BSS)?

Bernard-Soulier syndrome (BSS) is a rare bleeding disorder associated with mutations in GP9, characterized by giant platelets and decreased platelet adhesion. GP9 antibodies offer valuable approaches for BSS research:

• Diagnostic applications:

  • Flow cytometry using anti-GP9 antibodies can quantify GP9 expression levels on patient platelets compared to healthy controls

  • Western blot analysis can detect altered protein size or expression levels resulting from mutations

• Mutation-specific investigations:

  • For known mutations, custom antibodies targeting mutation-specific epitopes can be developed

  • Combination of wild-type and mutation-specific antibodies can help characterize the molecular phenotype

• Structure-function relationship studies:

  • Immunoprecipitation with GP9 antibodies followed by mass spectrometry can identify altered protein interactions in BSS patients

  • Confocal microscopy with fluorescently-labeled GP9 antibodies can reveal abnormal membrane localization

• Therapeutic development evaluation:

  • GP9 antibodies can monitor restoration of GP9 expression in experimental therapies

  • Flow cytometry with GP9 antibodies can assess platelet function following intervention

This multifaceted approach provides comprehensive insights into BSS pathophysiology and potential therapeutic strategies, leveraging both polyclonal antibodies for broad detection and monoclonal antibodies for specific epitope recognition .

What methods can be used to study GP9 glycosylation patterns using available antibodies?

GP9 glycosylation is crucial for protein function, and specialized methods can elucidate these patterns:

• Glycosidase treatment coupled with Western blot:

  • Treat samples with enzymes like PNGase F (removes all N-linked glycans) or Endo-D (removes specific glycan structures)

  • Compare molecular weight shifts before and after treatment using Western blot with GP9 antibodies

  • The difference in migration patterns reveals the extent of glycosylation

• Lectin affinity analysis:

  • Perform immunoprecipitation with GP9 antibodies

  • Probe with different lectins that recognize specific glycan structures

  • This approach reveals the types of glycans present on GP9

• Mass spectrometry glycoprofiling:

  • Immunopurify GP9 using available antibodies

  • Perform glycopeptide analysis by mass spectrometry

  • This provides detailed structural information about glycan composition

• Glycan-specific antibody complementation:

  • Use antibodies that recognize specific glycan structures (such as Man5GlcNAc2) alongside GP9 antibodies

  • Co-localization studies reveal the presence of specific glycan structures on GP9

Research has shown that glycan structures like Man5GlcNAc2 are important in GP9 interactions, similar to how they function in other glycoproteins like HIV envelope glycoproteins . This understanding can inform studies on GP9 glycosylation patterns in normal and pathological states.

How can researchers investigate GP9's role in the GPIb-IX-V complex using available antibodies?

The GPIb-IX-V complex is critical for platelet adhesion and activation, with GP9 serving as an essential component. To investigate GP9's role within this complex:

• Co-immunoprecipitation studies:

  • Use GP9 antibodies to pull down the complex

  • Western blot with antibodies against other components (GPIbα, GPIbβ, GPV)

  • This approach identifies protein-protein interactions and complex integrity

• Proximity ligation assay (PLA):

  • Apply GP9 antibodies alongside antibodies against other complex components

  • PLA signals indicate close proximity (<40 nm) between proteins

  • This technique visualizes intact complexes in situ

• Functional blocking experiments:

  • Apply GP9 antibodies to platelets before functional assays

  • Measure effects on von Willebrand factor binding or platelet aggregation

  • This determines which GP9 epitopes are functionally critical

• Protein cross-linking studies:

  • Cross-link proteins in intact platelets

  • Immunoprecipitate with GP9 antibodies

  • Mass spectrometry analysis identifies cross-linked partners

  • This approach maps the three-dimensional architecture of the complex

• Super-resolution microscopy:

  • Label GP9 and other complex components with antibodies conjugated to different fluorophores

  • Techniques like STORM or PALM provide nanoscale resolution of complex organization

  • This reveals spatial relationships between components

These methodologies provide comprehensive insights into how GP9 contributes to complex assembly, stability, and function in both normal and pathological conditions .

What are the common challenges when working with GP9 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with GP9 antibodies:

• Low signal intensity:

  • Cause: Insufficient antibody concentration or low GP9 expression

  • Solution: Increase antibody concentration within recommended ranges (e.g., 1:100 instead of 1:500 for Western blot) ; use signal amplification systems like tyramide signal amplification; enrich platelets in samples

• High background:

  • Cause: Non-specific binding or excessive antibody concentration

  • Solution: Optimize blocking (try different blockers like BSA vs. milk); increase washing steps; titrate antibody to lower concentration; use more specific monoclonal antibodies when possible

• Cross-reactivity issues:

  • Cause: Antibody recognizing proteins similar to GP9

  • Solution: Validate with appropriate controls; consider using monoclonal antibodies with defined epitope specificity; perform peptide competition assays to confirm specificity

• Sample degradation:

  • Cause: GP9 protein degradation during preparation

  • Solution: Use fresh samples; add protease inhibitors; maintain cold chain; avoid freeze-thaw cycles; optimize lysis buffers for membrane proteins

• Inconsistent results between experiments:

  • Cause: Batch-to-batch antibody variability (especially with polyclonals)

  • Solution: Use monoclonal antibodies for better consistency ; purchase larger lots of antibody when possible; standardize protocols rigorously

• Glycosylation interference:

  • Cause: Variable glycosylation affecting epitope accessibility

  • Solution: Use antibodies targeting protein backbone rather than glycan-modified regions; consider deglycosylation treatments before analysis

Addressing these challenges through methodical optimization improves reproducibility and data quality in GP9 research.

How should researchers optimize storage and handling of GP9 antibodies to maintain performance?

Proper storage and handling are critical for maintaining antibody performance over time:

• Storage temperature considerations:

  • Unconjugated antibodies: Store at -20°C for long-term storage or at 4°C for up to one month

  • Conjugated antibodies (e.g., APC-conjugated): Store at 4°C protected from light

  • Avoid storing at room temperature for any extended period

• Aliquoting strategy:

  • Upon receipt, divide antibody into small single-use aliquots

  • Use low-binding microcentrifuge tubes to prevent protein loss

  • Label aliquots with date, concentration, and number of freeze-thaw cycles

• Freeze-thaw cycle management:

  • Minimize freeze-thaw cycles, as each cycle can reduce activity by 5-10%

  • Thaw antibodies slowly on ice rather than at room temperature

  • Never use heat to thaw antibodies

• Buffer considerations:

  • Most GP9 antibodies are supplied in PBS containing preservatives like 0.09% sodium azide

  • Some contain glycerol (up to 50%) as a cryoprotectant

  • Do not dilute stock antibody unless immediately using

• Contamination prevention:

  • Use sterile technique when handling antibodies

  • Avoid introducing bacteria or fungi which can degrade antibodies

  • Include sodium azide (0.02%) in working solutions for microbial inhibition

• Transportation requirements:

  • Maintain cold chain during transportation

  • Use ice packs or dry ice depending on shipping duration

  • Some antibodies require temperature-controlled shipping and are not eligible for return due to quality concerns

Following these guidelines helps maintain antibody performance throughout its shelf life and ensures consistent experimental results.

What controls should be included when using GP9 antibodies for different applications?

Appropriate controls are essential for result interpretation and validation:

For Western Blot:

• Positive control: K-562 cell lysate has been validated as a positive control for GP9 detection

• Negative control: Cell lines known not to express GP9

• Loading control: Probe for housekeeping proteins (β-actin, GAPDH) to normalize expression

• Molecular weight marker: Confirm the expected 19 kDa size of GP9

• Primary antibody omission: Detect non-specific binding of secondary antibody

• Isotype control: Use matching isotype antibody (IgG for polyclonals, IgG1 or IgG2a for monoclonals) to assess background

For Flow Cytometry:

• Unstained control: Establish autofluorescence baseline

• Isotype control: Match antibody class and conjugate (e.g., Mouse IgG2a-APC for GR-P clone)

• Single-stained controls: Essential for compensation in multicolor experiments

• Fluorescence-minus-one (FMO) controls: Determine proper gating boundaries

• Known positive samples: Confirm staining protocol works effectively

For Immunohistochemistry:

• Tissue with known GP9 expression: Validate staining pattern

• Antibody omission: Assess endogenous peroxidase activity

• Blocking peptide competition: Confirm specificity of staining

• Isotype control: Match concentration and species of primary antibody

For ELISA:

• Standard curve: Use recombinant GP9 protein for quantitation

• Blank wells: Assess background from reagents

• Known concentration samples: Validate assay accuracy

Including these controls enables confident interpretation of results and helps troubleshoot any unexpected outcomes across different experimental platforms.

How might GP9 antibodies contribute to research on novel antiplatelet therapies?

GP9 antibodies offer significant potential for developing and evaluating novel antiplatelet therapies:

• Therapeutic target screening:

  • GP9 antibodies can help identify compounds that modulate GP9 expression or function

  • High-throughput screening assays using flow cytometry with GP9 antibodies can measure effects of compound libraries on GP9 expression

• Mechanism of action studies:

  • Monitor conformational changes in the GPIb-IX-V complex using conformation-specific GP9 antibodies

  • Investigate whether potential therapeutics alter GP9 glycosylation or complex assembly

• Drug delivery system development:

  • GP9 antibodies can be utilized to develop platelet-targeted drug delivery systems

  • Conjugation of therapeutic compounds to GP9-targeting fragments can provide platelet specificity

• Biomarker development:

  • GP9 antibodies enable quantification of platelet surface GP9 as a potential biomarker for antiplatelet therapy efficacy

  • Flow cytometry protocols with standardized GP9 antibodies could become clinical tools for therapy monitoring

• Patient stratification approaches:

  • GP9 antibody-based assays might identify patient subgroups more likely to respond to specific antiplatelet therapies

  • Correlate GP9 expression patterns with clinical outcomes to develop personalized treatment protocols

• Off-target effect assessment:

  • Evaluate whether novel therapeutics unintentionally affect GP9 expression or function

  • Develop safety screening protocols incorporating GP9 antibodies

This research direction has significant clinical translation potential, especially for developing therapies for conditions where current antiplatelet drugs show limitations or unacceptable side effects.

What emerging technologies could enhance GP9 antibody-based research?

Several cutting-edge technologies show promise for advancing GP9 antibody applications:

• Mass cytometry (CyTOF):

  • Combines flow cytometry with mass spectrometry

  • Allows simultaneous measurement of >40 parameters

  • Metal-conjugated GP9 antibodies enable highly multiplexed analysis of platelet protein networks

  • Provides deeper insights into GPIb-IX-V complex regulation in different physiological states

• Single-cell proteomics:

  • Combines GP9 antibodies with single-cell isolation techniques

  • Reveals heterogeneity in platelet populations regarding GP9 expression

  • Correlates GP9 levels with other platelet proteins at single-cell resolution

• Super-resolution microscopy:

  • Techniques like STORM, PALM, and STED overcome the diffraction limit

  • Fluorophore-conjugated GP9 antibodies enable nanoscale visualization of GPIb-IX-V complex organization

  • Reveals spatial relationships between GP9 and other membrane components

• Automated microfluidic platforms:

  • Combine GP9 antibody detection with controlled flow conditions

  • Real-time monitoring of platelet adhesion and aggregation under physiological flow

  • High-throughput screening of compounds affecting GP9 function

• CRISPR-Cas9 combined with GP9 antibody detection:

  • Create precise GP9 mutations or regulatory element modifications

  • Measure effects on expression and function using GP9 antibodies

  • Provides insights into structure-function relationships

• Humanized GP9 antibodies for in vivo studies:

  • Develop therapeutic-grade humanized versions of research antibodies

  • Enable translational studies with reduced immunogenicity

  • Bridge the gap between basic research and clinical applications

These emerging technologies will significantly enhance our understanding of GP9 biology and accelerate development of GP9-targeted therapeutics for platelet-related disorders.

How can researchers integrate GP9 antibody data with other -omics approaches for comprehensive platelet studies?

Integration of GP9 antibody data with multi-omics approaches provides a systems biology perspective on platelet function:

• Proteogenomic integration:

  • Correlate GP9 protein levels (measured by antibodies) with GP9 gene expression

  • Identify post-transcriptional mechanisms regulating GP9 expression

  • Map genetic variants affecting GP9 expression or function

• Glycoproteomic analysis:

  • Combine GP9 antibody-based quantification with glycan structural analysis

  • Immunoprecipitate GP9 using antibodies followed by glycan characterization

  • Correlate glycosylation patterns with protein function and stability

• Interactome mapping:

  • Use GP9 antibodies for co-immunoprecipitation followed by mass spectrometry

  • Identify novel GP9 interaction partners beyond known GPIb-IX-V components

  • Construct network maps of GP9-associated protein complexes

• Spatial transcriptomics with protein validation:

  • Correlate spatial distribution of GP9 mRNA with protein localization

  • Use GP9 antibodies to validate transcriptomic findings at protein level

  • Develop comprehensive maps of platelet protein distribution

• Multi-parameter data integration:

  • Develop computational pipelines integrating GP9 antibody data with genomic, transcriptomic, metabolomic datasets

  • Apply machine learning algorithms to identify patterns associated with platelet disorders

  • Create predictive models of platelet function based on integrated -omics data

• Temporal dynamics studies:

  • Monitor changes in GP9 expression and localization during platelet activation

  • Correlate with transcriptomic and metabolomic changes over time

  • Develop dynamic models of platelet activation incorporating GP9 regulation

This integrated approach provides unprecedented insights into platelet biology, potentially revealing novel therapeutic targets and diagnostic biomarkers for cardiovascular and hematological disorders.

What are the current best practices for GP9 antibody selection and validation?

Based on current research standards, these best practices ensure reliable GP9 antibody use:

• Selection criteria prioritization:

  • Match antibody type to application (polyclonal for greater sensitivity, monoclonal for specificity)

  • Verify validation data for your specific application (WB, FC, IHC)

  • Select antibodies with validation in multiple cell types/tissues

  • Check cross-reactivity data if working across species

• Comprehensive validation workflow:

  • Verify antibody performance in your specific system before full experiments

  • Implement at least two orthogonal techniques to confirm findings

  • Use genetic approaches (siRNA, CRISPR) to validate specificity when possible

  • Document detailed validation methods in publications

• Standardization approaches:

  • Maintain detailed antibody records including catalog number, lot, concentration

  • Establish standard operating procedures for each application

  • Use consistent positive controls across experiments

  • Implement quantitative metrics for antibody performance

• Reproducibility enhancement:

  • Report all antibody details in publications (following antibody reporting guidelines)

  • Share detailed protocols including optimization steps

  • Consider antibody authentication services for independent validation

  • Deposit validation data in public repositories when possible

These practices align with recent initiatives to improve antibody reliability in biomedical research, ensuring that GP9 antibody-based findings are robust and reproducible across laboratories.

What research gaps remain in our understanding of GP9 that antibody-based approaches could address?

Despite significant progress, several knowledge gaps remain where GP9 antibodies could provide crucial insights:

• Structural dynamics of the GPIb-IX-V complex:

  • Develop conformation-specific antibodies to capture different states of GP9

  • Study how GP9 conformation changes during platelet activation

  • Investigate how GP9 structurally contributes to complex stability

• GP9 post-translational modifications beyond glycosylation:

  • Generate modification-specific antibodies (phosphorylation, ubiquitination)

  • Map how these modifications regulate GP9 function

  • Determine enzymes responsible for these modifications

• GP9 involvement in non-canonical signaling pathways:

  • Use GP9 antibodies to isolate and characterize novel signaling complexes

  • Investigate GP9's role beyond hemostasis (inflammation, immune interactions)

  • Explore potential receptor functions beyond von Willebrand factor binding

• GP9 expression heterogeneity in platelet populations:

  • Apply single-cell antibody-based techniques to quantify GP9 variability

  • Correlate GP9 expression with platelet function and aging

  • Investigate whether distinct GP9 expression levels define functional platelet subsets

• GP9 mechanisms in disorders beyond Bernard-Soulier syndrome:

  • Examine GP9 expression/function in acquired platelet disorders

  • Investigate GP9's role in thromboinflammatory conditions

  • Explore potential GP9 alterations in metabolic disorders affecting platelets