TSPAN9 Antibody, HRP conjugated

What is TSPAN9 and what is its biological significance?

TSPAN9 (Tetraspanin-9) is a 239 amino acid multi-pass membrane protein belonging to the tetraspanin (TM4SF) family. Tetraspanins are cell surface proteins that regulate cell development, activation, growth, and motility through signal transduction. TSPAN9 contains four hydrophobic domains characteristic of tetraspanins . The protein is encoded by a gene that maps to human chromosome 12p13.33 .

TSPAN9 forms complexes with GPVI in tetraspanin microdomains on platelet surfaces, suggesting a role in platelet function . Recent research has identified TSPAN9 as a potential biomarker for colorectal cancer (CRC), where it demonstrated an area under the ROC curve (AUC) of 0.87 individually and 0.98 when combined with CD59 for distinguishing CRC patients from healthy controls . This makes TSPAN9 particularly valuable for early-stage (I and II) CRC detection.

The protein is also known by several synonyms including NET5, NET-5, PP1057, Tspan-9, and Tetraspan NET-5 . In subcellular localization studies, TSPAN9 has been identified in extracellular compartments .

Proper storage and handling of TSPAN9 Antibody, HRP conjugated is critical for maintaining its activity and specificity. Based on manufacturer recommendations:

The antibody should be stored at -20°C in aliquots to avoid repeated freeze-thaw cycles, which can damage the antibody and reduce its activity . The typical storage buffer consists of an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol .

When handling HRP-conjugated antibodies, researchers should be aware that byproducts of HRP-induced chemiluminescence are free radicals that can potentially inactivate the HRP itself (giving a visible yellow/brown color) and irreversibly damage the antibodies, proteins, and membrane . This characteristic is particularly important to consider when planning stripping and reprobing experiments.

For long-term stability, avoid exposure to direct light and keep the antibody in temperature-controlled environments during experimental procedures. Working aliquots should be prepared to minimize the need to thaw the entire stock repeatedly.

What controls should be included when using TSPAN9 Antibody, HRP conjugated?

Proper experimental controls are essential for validating results obtained with TSPAN9 Antibody, HRP conjugated:

Positive Controls:

• Cell lines or tissues known to express TSPAN9 (based on the search results, colorectal cancer cell lines would be appropriate)

• Recombinant TSPAN9 protein

Negative Controls:

• Samples from TSPAN9 knockout models (if available)

• Cell lines with confirmed low/no expression of TSPAN9

• Isotype control (rabbit IgG-HRP conjugated) at the same concentration as the primary antibody

Technical Controls:

• No primary antibody control to assess non-specific binding of detection systems

• Loading controls for Western blot (e.g., α-tubulin, as mentioned in search result )

• For IHC, include tissue known to be negative for TSPAN9

When detecting phosphorylated proteins alongside TSPAN9, researchers should consider the limitations of chemiluminescence detection with HRP conjugates, as signals from proteins of similar molecular weights cannot be resolved on the same membrane without stripping and reprobing .

How does TSPAN9 Antibody, HRP conjugated compare to fluorescent detection methods?

Choosing between HRP-conjugated and fluorescent detection methods involves several considerations:

Advantages of HRP-conjugated TSPAN9 Antibody:

• Lower cost compared to fluorescent detection systems

• Higher sensitivity for low abundance proteins

• Compatible with standard chemiluminescence imaging equipment

• Signal amplification through enzymatic reaction

Limitations of HRP-conjugated detection:

• Cannot be used for multiplexed detection of targets with similar molecular weights without stripping and reprobing

• The byproducts of HRP-induced chemiluminescence are free radicals that can inactivate the HRP itself and irreversibly damage antibodies, proteins, and membranes

• Limited dynamic range compared to some fluorescent methods

Fluorescent detection advantages:

• Permits interrogation of multiple targets from the same sample while avoiding stripping/reprobing processes

• Better for quantitative analysis due to wider dynamic range

• Signals remain stable over time, allowing for re-imaging

What methodologies optimize TSPAN9 detection in extracellular vesicles (EVs)?

TSPAN9 has emerged as a significant biomarker in extracellular vesicles, particularly in colorectal cancer research. Based on the literature, researchers have developed optimized protocols for TSPAN9 detection in EVs:

Isolation of EVs:

• Collect culture supernatants or plasma samples

• Perform differential ultracentrifugation (typically 100,000-120,000× g)

• Validate EV isolation by size measurement (target range: ~97-107 nm) and confirmation of EV markers such as CD9

TSPAN9 Quantification in EVs:
For precise quantification, targeted mass spectrometry using product ion scanning (PIS) mode has demonstrated high precision with median CV values below 20% . The quantifiable range for TSPAN9 in plasma-derived EVs has been reported in healthy controls and CRC patients as follows:

*Specific values not provided in the search results, but the study indicated significant elevation in CRC patients .

When analyzing TSPAN9 in EVs, researchers should consider including CD59 measurements, as the combination of TSPAN9 and CD59 has shown excellent diagnostic potential for CRC with an AUC of 0.98, significantly better than either marker alone .

How can researchers troubleshoot non-specific binding or weak signals when using TSPAN9 Antibody, HRP conjugated?

When encountering issues with TSPAN9 Antibody, HRP conjugated, consider the following troubleshooting approaches:

For weak or no signal:

• Antibody concentration: Increase the antibody concentration within the recommended range (1:300-5000 for WB, 1:200-400 for IHC-P)

• Antigen retrieval: For IHC-P, optimize antigen retrieval methods to ensure proper exposure of the epitope

• Protein denaturation: Ensure complete denaturation of samples for Western blot

• HRP activity: Verify the activity of HRP using a test substrate; HRP can be inactivated by improper storage or handling

• Detection reagents: Use fresh ECL substrate with appropriate sensitivity range

For non-specific binding:

• Blocking optimization: Increase blocking time or concentration of blocking agent

• Antibody specificity: Verify the epitope location (the antibody targets region 131-239 of the 239 amino acid protein)

• Washing stringency: Increase washing steps or detergent concentration

• Cross-reactivity: Check for potential cross-reactivity with related tetraspanin family members

For high background:

• Reduce antibody concentration: Dilute the antibody further within recommended range

• Extend washing: Increase washing duration and number of washes

• Buffer optimization: Adjust detergent concentration in washing buffers

• HRP byproducts: Be aware that HRP byproducts can cause yellowish-brown discoloration and damage to the membrane

When troubleshooting, make one change at a time and document results carefully to identify the optimal conditions for your specific experimental setup.

What are the considerations for quantitative analysis of TSPAN9 expression using HRP-conjugated antibodies?

Quantitative analysis of TSPAN9 expression using HRP-conjugated antibodies requires careful attention to several factors:

Signal Linearity and Dynamic Range:

• HRP chemiluminescence has a narrower dynamic range compared to fluorescent detection

• Perform titration experiments to establish the linear range of detection for your samples

• Use multiple exposure times to ensure signals fall within the linear range of detection

Normalization Strategy:

• For Western blot, normalize TSPAN9 signals to appropriate loading controls (e.g., α-tubulin, β-actin)

• For IHC-P, use appropriate tissue controls and standardized scoring systems

• When comparing different samples, consider using a standard curve of recombinant TSPAN9 protein

Image Acquisition and Analysis:

• Ensure consistent exposure settings between experimental groups

• Use digital image analysis software that can accurately quantify band intensity or staining patterns

• Be cautious of signal saturation, which compromises quantitative accuracy

Challenges with Stripping and Reprobing:

• HRP byproducts can irreversibly damage proteins and membranes, affecting subsequent detection after stripping

• For truly quantitative comparisons between TSPAN9 and other proteins, consider using parallel blots or fluorescent detection instead

For absolute quantification of TSPAN9 in complex samples like plasma-derived EVs, targeted mass spectrometry approaches have demonstrated superior precision and sensitivity compared to antibody-based methods .

How can TSPAN9 Antibody be used in studying colorectal cancer biomarkers?

TSPAN9 has emerged as a promising biomarker for colorectal cancer (CRC), particularly when analyzing extracellular vesicles (EVs). Recent research provides specific methodologies for utilizing TSPAN9 Antibody in CRC biomarker studies:

EV-based TSPAN9 Analysis in CRC:
TSPAN9 in plasma-derived EVs has demonstrated excellent diagnostic potential for CRC, with an area under the ROC curve (AUC) of 0.87 individually . When combined with CD59, the diagnostic accuracy increases to an AUC of 0.98 . Most notably, these markers proved to be outstanding diagnostic markers for early-stage (I and II) CRC patients, with an AUC of 0.99 in tandem and an individual AUC of 0.86 for TSPAN9 .

Recommended Workflow for CRC Biomarker Studies:

• Isolate EVs from plasma samples using differential ultracentrifugation

• Validate EV isolation by size measurement and confirmation of EV markers (CD9)

• Process EVs for protein analysis (either by Western blot or mass spectrometry)

• Quantify TSPAN9 levels using HRP-conjugated antibodies or targeted mass spectrometry

• Consider parallel analysis of CD59 for improved diagnostic accuracy

Comparative Analysis Approach:
For comparative studies between healthy controls and CRC patients, research has demonstrated that:

• TSPAN9 is detectable in plasma-derived EVs from both healthy controls and CRC patients

• TSPAN9 levels are significantly elevated in CRC patients compared to healthy controls

• TSPAN9 can be detected in early-stage CRC, making it valuable for early detection strategies

This approach has been validated in clinical samples from 80 healthy controls and 73 CRC patients, demonstrating robust diagnostic potential .

What are the advantages and limitations of using different detection systems with TSPAN9 Antibody?

When working with TSPAN9 Antibody, researchers can choose between different detection systems, each with distinct advantages and limitations:

How does HRP conjugation affect the performance characteristics of TSPAN9 antibody?

HRP (horseradish peroxidase) conjugation directly impacts several aspects of TSPAN9 antibody performance:

Impact on Sensitivity and Detection Limit:
HRP conjugation provides enzymatic signal amplification, where a single HRP molecule can catalyze multiple reactions with the substrate, enhancing signal output. This characteristic makes HRP-conjugated antibodies particularly useful for detecting low-abundance proteins like TSPAN9 in certain samples.

Effects on Epitope Recognition:
The conjugation process can potentially affect antibody binding if the chemical conjugation occurs near the antigen-binding site. For TSPAN9 Antibody, the binding epitope is within the region 131-239 of the 239 amino acid protein , and careful conjugation protocols help maintain epitope recognition capacity.

Multiplexing Limitations:
HRP-conjugated antibodies have significant limitations for multiplexed detection:

• Cannot distinguish between proteins of similar molecular weights on the same membrane

• Require stripping and reprobing for detection of multiple targets, which can damage proteins and reduce sensitivity

• Byproducts of HRP-induced chemiluminescence are free radicals that can inactivate the HRP itself and irreversibly damage the antibodies, proteins, and membrane

Storage Stability Considerations:
HRP-conjugated antibodies typically contain 50% glycerol in their storage buffer to maintain stability . Researchers should be aware that HRP activity can diminish over time, particularly with repeated freeze-thaw cycles, making proper aliquoting and storage at -20°C or -80°C essential .

What is the specificity profile of commercially available TSPAN9 Antibodies, HRP conjugated?

Understanding the specificity profile of TSPAN9 Antibody, HRP conjugated is critical for experimental design and data interpretation:

Species Cross-Reactivity:
Commercial TSPAN9 Antibodies, HRP conjugated, show varying patterns of species reactivity:

Epitope Specificity:
The antibodies are typically raised against defined regions of the TSPAN9 protein:

• Bioss antibody: Targets amino acids 131-239 of the 239 amino acid human TSPAN9 protein

• AFG Scientific: Targets amino acids 107-203 of human Tetraspanin-9 protein

Potential Cross-Reactivity:
Tetraspanins share structural similarities that could potentially lead to cross-reactivity. The antibodies are generally validated against specific targets, but researchers should be aware of potential cross-reactivity with other tetraspanin family members, particularly in high-expression systems.

Validation Methods:
Commercial antibodies undergo validation using various methods, though specific validation data for each product may vary:

• Western blot against recombinant protein or cell/tissue lysates

• IHC-P on human tissues

• ELISA for binding capacity assessment

For critical experiments, particularly those involving novel tissue types or experimental conditions, researchers should consider performing additional validation using positive and negative controls appropriate for their specific application.

What are the recommended experimental protocols for using TSPAN9 Antibody, HRP conjugated in different applications?

Different applications require specific protocols for optimal results with TSPAN9 Antibody, HRP conjugated:

Western Blot Protocol:

• Sample preparation: Lyse cells/tissues in appropriate buffer containing protease inhibitors

• Protein quantification: Normalize loading to 20-50 μg total protein per lane

• SDS-PAGE: Separate proteins using 10-12% gels

• Transfer: Use PVDF or nitrocellulose membranes

• Blocking: 5% non-fat milk or BSA in TBST, 1 hour at room temperature

• Primary antibody: Dilute TSPAN9 Antibody, HRP conjugated 1:300-5000 in blocking buffer

• Incubation: Overnight at 4°C or 2 hours at room temperature

• Washing: 3-5× with TBST, 5-10 minutes each

• Detection: Apply ECL substrate directly (no secondary antibody needed)

• Imaging: Expose to X-ray film or use digital imaging system

Immunohistochemistry Protocol:

• Sample preparation: Formalin-fixed, paraffin-embedded sections (4-6 μm)

• Deparaffinization and rehydration

• Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes

• Protein blocking: 5-10% normal serum, 30 minutes

• Primary antibody: Dilute TSPAN9 Antibody, HRP conjugated 1:200-400

• Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Washing: 3× with PBS, 5 minutes each

• Detection: Apply DAB substrate directly (no secondary antibody needed)

• Counterstain, dehydrate, clear, and mount

ELISA Protocol:

• Coating: Capture antibody or target antigen at 1-10 μg/ml in coating buffer

• Blocking: 1-5% BSA in PBS, 1-2 hours at room temperature

• Sample addition: Add samples/standards in appropriate diluent

• Detection: Add TSPAN9 Antibody, HRP conjugated at optimized dilution

• Incubation: 1-2 hours at room temperature

• Washing: 4-5× with PBST

• Substrate addition: TMB substrate

• Stop reaction: Add stop solution after color development

• Read absorbance at 450 nm

For all applications, researchers should optimize antibody dilutions and incubation conditions for their specific experimental setup through preliminary titration experiments.

How can researchers validate the specificity of TSPAN9 Antibody, HRP conjugated in their experimental system?

Validating antibody specificity is crucial for generating reliable data. For TSPAN9 Antibody, HRP conjugated, consider these validation approaches:

Genetic Validation Approaches:

• TSPAN9 Knockdown/Knockout: Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate TSPAN9 expression, then confirm signal reduction with the antibody

• Overexpression: Compare signal in cells with normal versus overexpressed TSPAN9 levels

• Tagged TSPAN9: Express tagged TSPAN9 and compare detection with both anti-tag antibody and TSPAN9 antibody

Biochemical Validation Methods:

• Peptide Competition: Pre-incubate the antibody with the immunizing peptide (amino acids 131-239 or 107-203 depending on manufacturer) to block specific binding

• Size Verification: Confirm that the detected band matches the expected molecular weight of TSPAN9 (approximately 25-30 kDa)

• Multiple Antibodies: Compare results using antibodies targeting different epitopes of TSPAN9

Technical Validation Controls:

• Positive Control Tissues: Use tissues known to express TSPAN9 (e.g., platelets or colorectal cancer samples)

• Negative Control Tissues: Use tissues with minimal TSPAN9 expression

• Isotype Control: Use rabbit IgG-HRP at the same concentration to assess non-specific binding

Orthogonal Validation:

• mRNA Expression: Compare antibody signal with TSPAN9 mRNA levels by qPCR

• Mass Spectrometry: Confirm protein identity through targeted mass spectrometry

• Multi-method Detection: Compare results across different detection methods (e.g., immunofluorescence, flow cytometry)

Thorough validation not only confirms antibody specificity but also provides valuable controls for troubleshooting if experimental results become inconsistent over time.

What considerations should be made when designing experiments to study TSPAN9 interaction with other proteins?

TSPAN9 forms complexes with other proteins in tetraspanin microdomains, making interaction studies particularly relevant. When designing such experiments, consider:

Preservation of Membrane Protein Interactions:

• Lysis Conditions: Use mild detergents (e.g., CHAPS, Brij series) that preserve tetraspanin-enriched microdomains

• Sample Handling: Minimize temperature fluctuations and processing time to prevent complex dissociation

• Crosslinking: Consider reversible crosslinking approaches to stabilize transient interactions

Interaction Detection Methods:

• Co-immunoprecipitation: Pull down TSPAN9 and identify interacting partners

  • Note: HRP-conjugated antibodies are not ideal for immunoprecipitation; use unconjugated versions

• Proximity Ligation Assay: Detect in situ protein interactions with high specificity

• FRET/BRET: For live-cell analysis of dynamic interactions

Functional Validation of Interactions:

• Domain Mapping: Identify specific domains of TSPAN9 involved in protein interactions

• Mutational Analysis: Create point mutations to disrupt specific interactions

• Functional Assays: Assess the impact of disrupting TSPAN9 interactions on cellular functions

Special Considerations for TSPAN9:

• GPVI Interaction: TSPAN9 forms a complex with GPVI in tetraspanin microdomains on the platelet surface

• Extracellular Vesicle Localization: Consider how interactions may differ in EVs versus cellular membranes

• Cancer Context: Examine how TSPAN9 interactions may be altered in cancer cells, particularly in colorectal cancer where TSPAN9 has diagnostic value

When reporting interaction data, researchers should clearly describe experimental conditions, particularly detergent types and concentrations, as these significantly impact tetraspanin complex preservation and detection sensitivity.