TSPAN9 Antibody, Biotin conjugated

What is TSPAN9 and what cellular processes is it involved in?

TSPAN9 (Tetraspanin 9, also known as NET-5) is a 239 amino acid multi-pass membrane protein belonging to the tetraspanin (TM4SF) family. Tetraspanins regulate cell development, activation, growth, and motility through participation in signal transduction pathways. TSPAN9 specifically forms complexes with GPVI in tetraspanin microdomains on platelet surfaces. The gene encoding TSPAN9 maps to human chromosome 12p13.33, a region associated with various developmental disorders . As a member of the tetraspanin family, TSPAN9 contains four hydrophobic domains that facilitate its integration into the cell membrane and its participation in protein-protein interactions that regulate cellular functions .

What are the standard applications for TSPAN9 antibodies in research?

TSPAN9 antibodies are versatile research tools applicable to multiple experimental techniques:

• Western Blotting (WB): Recommended dilution 1:300-5000

• Flow Cytometry (FACS): Recommended dilution 1:20-100

• Immunohistochemistry (Paraffin-embedded Sections) (IHC-P): Recommended dilution 1:200-400

• Immunofluorescence (Paraffin-embedded Sections) (IF-P): Recommended dilution 1:50-200

These applications enable researchers to investigate TSPAN9 expression, localization, and interactions in various experimental models and clinical samples.

How should biotin-conjugated TSPAN9 antibodies be stored and handled for optimal performance?

Biotin-conjugated TSPAN9 antibodies require specific storage conditions to maintain their functionality:

• Shipping condition: 4°C

• Long-term storage: -20°C for up to one year

• Buffer composition: 0.01M TBS (pH 7.4) with 1% BSA, 0.02% ProClin300, and 50% Glycerol

• Avoid repeated freeze/thaw cycles as this may compromise antibody stability and performance

• ProClin preservative is classified as hazardous and should be handled by trained personnel only

Following these guidelines ensures maximum antibody stability and consistent experimental results.

What is the difference between unconjugated and biotin-conjugated TSPAN9 antibodies?

The primary difference lies in their detection capabilities:

The biotin-conjugated version offers advantages in signal amplification through the strong biotin-streptavidin interaction, which enables more sensitive detection in various applications .

How does the biotin conjugation affect the binding specificity and sensitivity of the TSPAN9 antibody?

• The biotin molecule's position may potentially affect antigen recognition if conjugated near the antigen-binding site

• Optimal biotin-to-antibody ratio must be maintained to prevent over-conjugation that could compromise binding

• While binding specificity is preserved (human, mouse, and rat reactivity is maintained), sensitivity can be enhanced due to signal amplification via the biotin-streptavidin system

When selecting between different conjugates (AbBy Fluor® dyes vs. biotin), researchers should consider the specific detection requirements of their experimental system and the potential for endogenous biotin interference.

What strategies can be employed to validate the specificity of biotin-conjugated TSPAN9 antibody staining?

To ensure experimental rigor, multiple validation approaches should be employed:

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide (KLH-conjugated synthetic peptide derived from human TSPAN9) to verify signal elimination

• Positive and negative control samples: Include tissues/cells known to express or lack TSPAN9

• Parallel detection methods: Compare results with unconjugated TSPAN9 antibodies targeting different epitopes

• siRNA knockdown validation: Confirm reduced signal in TSPAN9-knockdown samples

• Biotin blocking: Use avidin/biotin blocking kits to prevent endogenous biotin interference when working with biotin-rich samples

These validation steps are critical for confident interpretation of experimental findings, especially in complex tissues where non-specific binding may occur.

How can biotin-conjugated TSPAN9 antibodies be incorporated into multi-protein detection systems?

Biotin-conjugated TSPAN9 antibodies are valuable components in multi-protein detection strategies:

• Multiplexed immunofluorescence: Combine with other non-biotin primary antibodies followed by streptavidin conjugates with distinct fluorophores

• Chemical conjugation approaches: The biotin functionality can be leveraged in click chemistry approaches for building complex detection systems

• Three-protein conjugate generation: As demonstrated in checkpoint inhibitory T cell engagers (CiTEs), biotin-functionalized three-protein conjugates can be constructed using chemical methods like SPAAC or SPIEDAC reactions

• Proximity ligation assays: Detect TSPAN9 interactions with other proteins using biotin-conjugated antibodies combined with oligonucleotide-coupled streptavidin

For example, researchers developing multi-protein constructs can utilize the dibromopyridazinedione (Br₂PD) scaffold approach with bioorthogonal click handles to create sophisticated detection systems that incorporate biotin-conjugated TSPAN9 antibodies .

What considerations are critical when using biotin-conjugated TSPAN9 antibodies in flow cytometry versus immunohistochemistry?

Different applications require specific optimization strategies:

Flow Cytometry (FACS):

• Optimal dilution: 1:20-100

• Cell permeabilization is necessary for detecting internal epitopes

• Streptavidin conjugate selection affects sensitivity and resolution

• Compensation settings must account for streptavidin fluorophore characteristics

• Live/dead discrimination is essential to prevent non-specific binding to dead cells

Immunohistochemistry (IHC-P):

• Optimal dilution: 1:200-400

• Antigen retrieval methods may affect epitope accessibility

• Endogenous biotin blocking is critical for biotin-rich tissues (liver, kidney)

• Streptavidin-HRP or -AP systems provide flexible visualization options

• The fixation method significantly impacts epitope preservation and detection sensitivity

These application-specific considerations help researchers optimize their experimental protocols for maximum specificity and sensitivity.

How can researchers troubleshoot inconsistent results when using biotin-conjugated TSPAN9 antibodies?

When encountering variability in experimental outcomes, systematic troubleshooting should address:

• Antibody integrity: Evaluate for potential degradation due to improper storage or handling

• Sample preparation: Optimize fixation and permeabilization protocols for the specific sample type

• Blocking efficiency: Enhance blocking steps to reduce non-specific binding

• Endogenous biotin interference: Implement avidin/biotin blocking systems for biotin-rich tissues

• Detection system sensitivity: Adjust streptavidin conjugate concentration or incubation time

• Epitope masking: The AA 180-203 region might be inaccessible in certain experimental conditions; alternative fixation or antigen retrieval methods may be required

Systematic adjustment of these parameters should be documented to establish optimized protocols for specific experimental systems.

What chemical conjugation strategies can be employed for custom TSPAN9 antibody-biotin preparation?

For researchers requiring customized biotin conjugation to TSPAN9 antibodies:

• SPAAC (Strain-Promoted Azide-Alkyne Cycloaddition): Utilize bicyclononyne (BCN) strained alkyne-functionalized molecules to react with azide-modified antibodies

• SPIEDAC (Strain-Promoted Inverse Electron Demand Diels-Alder Cycloaddition): Employ tetrazine-BCN reactions for efficient biotin conjugation

• Dibromopyridazinedione (Br₂PD) scaffold: This approach enables re-bridging of disulfide bonds while incorporating bioorthogonal click handles

• Sequential one-pot reactions: Combine SPAAC with DBCO-biotin to create multi-functional conjugates

These chemical biology approaches allow for precise control over conjugation sites and stoichiometry, potentially enhancing antibody performance in specific applications.

How might biotin-conjugated TSPAN9 antibodies contribute to understanding tetraspanin biology in disease contexts?

The versatility of biotin-conjugated TSPAN9 antibodies positions them as valuable tools for investigating tetraspanin biology in various disease states:

• Platelet function disorders: Given TSPAN9's role in GPVI complexes on platelets, these antibodies can help elucidate mechanisms of platelet activation and aggregation abnormalities

• Cancer research: Tetraspanins often have altered expression in malignancies; biotin-conjugated TSPAN9 antibodies can be incorporated into multi-marker panels for tumor characterization

• Immunotherapy research: Building on CiTE technologies, TSPAN9-targeting constructs could potentially be developed using biotin-streptavidin bridging approaches

• Developmental biology: Chromosome 12p13.33 associations with developmental disorders suggest potential roles for TSPAN9 in developmental processes that could be investigated using these antibodies

The continued refinement of chemical conjugation techniques will likely expand the utility of these antibodies in complex experimental systems and therapeutic development.