CD9 Antibody, HRP conjugated

What is CD9 and why are CD9 antibodies important in research?

CD9 is a 25.4 kDa cell surface glycoprotein belonging to the transmembrane 4 superfamily, also known as the tetraspanin family. These proteins contain four transmembrane domains and form multimeric complexes with other cell surface proteins. CD9 functions in numerous cellular processes including differentiation, adhesion, and signal transduction, while playing a critical role in suppressing cancer cell motility and metastasis .

CD9 antibodies are essential research tools because they enable detection and functional investigation of this protein across multiple experimental systems. CD9 has been implicated in:

• Regulation of cell adhesion and motility through integrin interactions

• Platelet activation and aggregation processes

• Myelination signaling in the peripheral nervous system

• Formation of paranodal junctions

• Self-antigen and recall antigen-induced T cell activation

Researchers rely on CD9 antibodies to interrogate these pathways in both normal physiology and disease states.

How does HRP conjugation enhance CD9 antibody functionality?

HRP (horseradish peroxidase) conjugation provides significant advantages for CD9 antibody applications through direct enzymatic detection capabilities. The principal methodological benefits include:

• Elimination of secondary antibody requirements: Direct detection without additional incubation steps, reducing experimental variability and background noise

• Enhanced sensitivity: HRP enzymatic amplification allows detection of low-abundance CD9 in complex samples

• Versatility across applications: Compatible with multiple detection systems including colorimetric, chemiluminescent, and fluorescent substrates

• Streamlined workflows: Reduced protocol time and complexity with fewer washing steps and reagents

For example, in ELISA applications, CD9 antibody, HRP conjugated can be used directly for detection at concentrations of approximately 0.2 μg/mL when paired with recombinant CD9 proteins that have been serially diluted, as demonstrated in validation studies .

What are the typical applications of CD9 antibody, HRP conjugated?

CD9 antibody, HRP conjugated has been validated for multiple research applications requiring sensitive detection of CD9 expression:

When designing experiments, researchers should note that CD9 antibody has been successfully employed to detect CD9 in specific contexts including:

• MCF-7 human breast cancer cells (positive control)

• U937 human histiocytic lymphoma cells (negative control)

• Exosomes from HT29 and COLO205 cell lines

What optimization strategies should be employed when using CD9 antibody, HRP conjugated in flow cytometry?

When optimizing CD9 antibody, HRP conjugated for flow cytometry, researchers should implement a systematic approach:

• Titration determination: Though the recommended dilution is 1:100 , perform a titration series (1:50 to 1:500) to determine optimal signal-to-noise ratio for your specific cell type

• Buffer composition optimization:

  • Use PBS (pH 7.3) containing 1-2% BSA or FBS to reduce non-specific binding

  • Include 0.1% sodium azide to prevent internalization during staining

  • For fixed cells, add 0.1% saponin for membrane permeabilization if intracellular CD9 detection is desired

• Incubation parameters:

  • Maintain consistent temperature (typically 4°C for surface staining)

  • Optimize incubation time (30-60 minutes is standard, but may require adjustment)

  • Protect from light to prevent HRP photobleaching

• Controls implementation:

  • Positive control: MCF-7 human breast cancer cells (known CD9 expressers)

  • Negative control: U937 human histiocytic lymphoma cells

  • Isotype control: Mouse IgG1 matched to the CD9 antibody's isotype

• Substrate selection:

  • For direct HRP visualization, optimize substrate concentration (TMB, DAB, or AEC)

  • For multicolor panels, consider tyramide signal amplification systems compatible with flow cytometry

Validation data indicates that CD9 surface expression can be reliably detected following this methodology, with expected CD9 localization to both cell surface and cytoplasm .

How can CD9 antibody, HRP conjugated be employed for extracellular vesicle (EV) detection and isolation?

CD9 antibody, HRP conjugated serves as a powerful tool for EV research through multiple methodological approaches:

A. ELISA-based EV detection:

• Conjugate CD9 antibody with an affinity tag at concentration of 0.05 μg/mL

• Incubate with culture media from cell lines (e.g., HT29, COLO205) or ultracentrifuge-enriched serum exosomes

• Detect using HRP-conjugated antibody at 0.2 μg/mL

• Perform assay on microplates pre-coated with anti-tag antibody

B. Column-based CD9-HPLC immunoaffinity chromatography:

• Develop anti-CD9 HPLC column using UltraLink hydrazide resin conjugated with CD9 antibody via hydrazide chemistry

• Use approximately 20 μg of antibody per milliliter of resin

• Oxidize antibody with sodium meta-periodate to generate aldehydes

• Incubate oxidized antibody with hydrazide resin on a centrifuge column

• Wash with coupling buffer, 1M NaCl, and PBS with 0.05% sodium azide

• Pack into a PEEK column (4.6 mm × 50 mm) with approximately 0.8 mL column volume

• Load serum sample (as little as 40 μL) onto the column and collect EV fraction

• Perform post-purification cleaning using 50 kDa MWCO filter to desalt, concentrate, and reduce co-eluting serum proteins

This method effectively isolates EVs from microscale serum volumes, enabling downstream proteomic analysis while minimizing contamination from blood proteins and lipoprotein particles .

What techniques can be used to investigate the CD9 interactome during infection processes?

The CD9 interactome can be comprehensively characterized during infection using proximity labeling approaches. The following methodology has been validated:

• Preparation stage:

  • Implement proximity labeling using a CD9 fusion construct

  • Culture cells expressing the labeled CD9 construct

  • Introduce infectious agents (e.g., bacterial strains) at varying time points

• Time-course analysis:

  • Collect samples at multiple time points (e.g., 30, 60, and 240 minutes post-infection)

  • Process samples for proteomic analysis via nano-LC-MS/MS

• Data analysis:

  • Identify proteins enriched at each time point

  • Analyze shared and unique proteins across timepoints

  • Categorize enriched proteins by cellular location and function

Research has revealed that the CD9 interactome is highly dynamic during bacterial infection, with:

• 30 mins post-infection: Minimal unique proteins

• 60 mins post-infection: 21 unique enriched proteins

• 240 mins post-infection: 210 unique enriched proteins

• 346 proteins shared across all timepoints (representing a core interactome)

KEGG pathway analysis of CD9-proximal proteins has identified involvement in multiple cellular processes, including adherens junctions, tight junctions, endocytosis, cell adhesion molecules, and SNARE interactions. Interestingly, proteins associated with several bacterial infection pathways have been identified even without infection challenge .

How can researchers validate the specificity of CD9 antibody, HRP conjugated in experimental systems?

Validating CD9 antibody specificity requires a multi-faceted approach:

• Cellular expression validation:

  • Compare staining patterns between known CD9-positive cells (e.g., MCF-7) and CD9-negative cells (e.g., U937)

  • Confirm expected subcellular localization (cell surface and cytoplasm for CD9)

• Molecular validation:

  • Perform Western blot analysis confirming single band at expected molecular weight (25.4 kDa for CD9)

  • Run CD9 knockdown/knockout controls to verify signal reduction

• Cross-reactivity assessment:

  • Test against cells from multiple species if performing cross-species experiments

  • Confirm reactivity with human, mouse, and rat samples if using antibodies validated for these species

• Functional validation:

  • Verify antibody function in immunoprecipitation experiments prior to proximity labeling studies

  • Confirm antibody's ability to detect CD9 in extracellular vesicles through comparative isolation methods

• Recombinant protein controls:

  • Perform ELISA with serially diluted recombinant CD9 protein (e.g., 10015-CD) to establish detection limits

  • Use pre-adsorption with recombinant protein to validate signal specificity

A comprehensive validation strategy ensures experimental results accurately reflect CD9 biology rather than non-specific binding or artifacts.

What methodological considerations are important when investigating CD9's role in cancer metastasis suppression?

When investigating CD9's role in cancer metastasis suppression, researchers should consider these methodological approaches:

• Expression analysis in cancer progression:

  • Use CD9 antibody, HRP conjugated in tissue microarrays to correlate CD9 expression levels with metastatic status

  • Compare primary tumors with matched metastatic lesions to track CD9 expression changes

  • Incorporate matched normal tissues as controls

• Functional assays:

  • Employ CD9 antibody in migration and invasion assays to assess functional blocking

  • Combine with CD9 overexpression or knockdown studies to validate antibody specificity

  • Use validated cell models such as MCF-7 (CD9-positive) breast cancer cells

• Interactome characterization:

  • Implement proximity labeling approaches to identify CD9 interaction partners in metastatic vs. non-metastatic cells

  • Focus on known CD9 interactors including:

    • Integrins (particularly α5 and β1)

    • Other tetraspanin members (CD151, Tspan15)

    • Immunoglobulin superfamily members (IGSF3)

• Pathway analysis:

  • Integrate CD9 interactome data with KEGG pathway analysis to identify:

    • Adherens junctions

    • Tight junctions

    • Endocytosis pathways

    • Cell adhesion molecules

• In vivo validation:

  • Utilize CD9 antibody for immunohistochemical validation in animal models of metastasis

  • Correlate changes in CD9 expression with metastatic burden

These methodologies provide a comprehensive framework for investigating the molecular mechanisms through which CD9 suppresses cancer cell motility and metastasis.

How can CD9 antibody, HRP conjugated be optimally stored and handled to maintain functionality?

To maintain optimal functionality of CD9 antibody, HRP conjugated, implement these evidence-based storage and handling protocols:

• Storage conditions:

  • Store at -20°C in manufacturer-provided buffer (typically PBS pH 7.3 containing 1% BSA, 50% glycerol)

  • Avoid repeated freeze-thaw cycles by aliquoting upon initial thaw

  • Track storage time, as stability is typically guaranteed for 12 months from receipt

• Working solution preparation:

  • Dilute in PBS (pH 7.3) immediately before use

  • Prepare fresh working solutions for each experiment

  • Calculate precise dilutions based on starting concentration (typically 0.5 mg/ml)

• Shipping and temporary storage:

  • Receive on blue ice or with ice packs

  • Transfer immediately to -20°C upon receipt

  • Avoid storage at 4°C for extended periods which can compromise HRP activity

• Preserving HRP activity:

  • Shield from direct light during all experimental procedures

  • Avoid exposure to heavy metals, oxidizing agents, and azides

  • Use freshly prepared substrates for detection

• Quality control monitoring:

  • Include positive controls in each experiment

  • Monitor signal intensity across experiments

  • Document lot-to-lot variations

These practices ensure maximum reproducibility and sensitivity across experiments while extending the functional lifespan of the antibody.

How should researchers integrate CD9 antibody data with other tetraspanin markers for comprehensive analysis?

Integrating CD9 data with other tetraspanin markers requires sophisticated analytical approaches:

• Tetraspanin co-expression analysis:

  • Design multiplex panels incorporating CD9 with related tetraspanins (CD151, Tspan15)

  • Account for spectral overlap when using multiple HRP-conjugated antibodies

  • Implement sequential detection protocols if using the same detection system

• Tetraspanin enrichment table for experimental planning:

• Integrated pathway analysis:

  • Combine CD9 interactome data with other tetraspanin datasets

  • Identify shared and unique pathway enrichments

  • Focus on convergent pathways such as:

    • Adherens junctions

    • Tight junctions

    • Endocytosis

    • Cell adhesion molecules

• Temporal dynamics consideration:

  • Analyze time-dependent changes in tetraspanin interactions

  • Compare dynamics across different tetraspanins during cellular processes

  • Document unique and shared temporal patterns

• Functional validation:

  • Design perturbation experiments targeting multiple tetraspanins

  • Assess combinatorial effects versus single tetraspanin manipulations

  • Validate findings with genetic approaches (siRNA, CRISPR)

This integrated approach provides a systems-level understanding of tetraspanin function while contextualizing CD9-specific findings within the broader tetraspanin network.

What considerations are critical when analyzing CD9-positive extracellular vesicles in biomarker studies?

When employing CD9 antibody, HRP conjugated for EV biomarker studies, researchers should consider these methodological factors:

• Sample preparation optimization:

  • Implement consistent pre-analytical procedures for all samples

  • Standardize starting volumes (as little as 40 μL serum has been validated)

  • Document sample collection-to-processing time

• Isolation method validation:

  • Compare CD9-HPLC immunoaffinity chromatography with ultracentrifugation methods

  • Assess recovery efficiency using spike-in standards

  • Characterize isolates by:

    • Transmission electron microscopy (TEM)

    • Nanoparticle tracking analysis (NTA)

    • Western blotting for EV markers

• Subpopulation characterization:

  • Determine if CD9-positive EVs represent all or subset of total EVs

  • Compare with other tetraspanin markers (CD63, CD81)

  • Account for tissue/cell-type specific differences in CD9 expression

• Purity assessment:

  • Implement 50 kDa MWCO filtration to reduce co-eluting serum proteins

  • Monitor contaminating lipoprotein particles

  • Quantify protein:particle ratios to assess preparation purity

• Downstream analysis optimization:

  • Validate proteomic workflows for CD9-isolated EVs

  • Establish minimum input requirements for reliable detection

  • Implement batch effect correction in longitudinal studies

These considerations ensure that CD9-positive EV analysis yields reproducible and biologically meaningful results in biomarker discovery and validation studies.

How might emerging technologies enhance CD9 antibody applications in future research?

Emerging technologies present exciting opportunities to expand CD9 antibody applications:

• Single-cell spatial proteomics:

  • Integration of CD9 antibody, HRP conjugated with spatial transcriptomics

  • Development of multiplexed imaging platforms for tetraspanin co-localization

  • Single-molecule tracking of CD9 dynamics in living cells

• Advanced proximity labeling approaches:

  • Expansion of CD9 interactome studies to broader infection models

  • Time-resolved proximity labeling with temporal resolution <15 minutes

  • Cell-type specific CD9 interactome mapping in complex tissues

• EV characterization innovations:

  • Microfluidic platforms for CD9-based EV isolation from limited samples

  • Combined CD9-capture and molecular profiling in single workflows

  • Integration of artificial intelligence for EV subpopulation classification

• Therapeutic applications:

  • Development of CD9-targeting approaches for cancer metastasis inhibition

  • Engineering CD9-positive EVs for therapeutic payload delivery

  • Creation of CD9-based diagnostic platforms for early disease detection

These technological advancements will continue to expand our understanding of CD9 biology while creating new opportunities for diagnostic and therapeutic applications.