TM9SF3 Antibody

What is TM9SF3 and what is its molecular structure?

TM9SF3 (transmembrane 9 superfamily member 3) is a member of the TM9SF family, also known as nonaspanins. The protein is characterized by a large noncytoplasmic domain and nine putative transmembrane domains . It has a calculated molecular weight of 68 kDa (589 amino acids), though the observed molecular weight can vary between 68-90 kDa depending on the experimental conditions and possibly post-translational modifications . TM9SF3 is also known by alternative names including SMBP and EP70-P-iso . The protein is encoded by the TM9SF3 gene (Gene ID: 56889) .

What are the standard specifications for TM9SF3 antibodies used in research?

Most commercially available TM9SF3 antibodies are polyclonal antibodies raised in rabbits against recombinant fusion proteins corresponding to human TM9SF3 . These antibodies typically:

• Have IgG isotype

• Are provided in liquid form

• Are purified by antigen affinity chromatography or similar methods

• Are stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Have recommended storage conditions of -20°C, with stability typically guaranteed for one year after shipment

• Show reactivity with human and mouse samples

What applications are TM9SF3 antibodies validated for?

Most commercially available TM9SF3 antibodies are validated primarily for Western Blot (WB) applications . The recommended dilution ranges for Western Blot typically fall between 1:500-1:2000 . Some antibodies may also be validated for ELISA applications . It is important to note that optimal dilutions should be determined by the end user for each specific experimental system, as sensitivity can vary between different tissue samples and experimental conditions .

How can researchers optimize Western Blot protocols for TM9SF3 detection?

For optimal TM9SF3 detection in Western Blot applications:

• Sample preparation: Since TM9SF3 is a transmembrane protein, ensure complete solubilization using appropriate lysis buffers containing detergents suited for membrane proteins.

• Protein loading: Load 20-40 μg of total protein per lane, with higher amounts recommended for tissues with lower expression levels.

• Antibody concentration: Start with a 1:1000 dilution for primary antibody incubation and optimize as needed based on signal-to-noise ratio .

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature.

• Detection method: Use enhanced chemiluminescence (ECL) with appropriate sensitivity for your expected expression level.

• Molecular weight considerations: Be aware that while calculated MW is 68 kDa, observed MW can vary (one source notes 90 kDa) , likely due to post-translational modifications or differences in gel systems.

• Positive control: Mouse stomach tissue has been confirmed as a positive control for TM9SF3 antibody validation .

What blocking and washing conditions optimize signal-to-noise ratio for TM9SF3 antibodies?

Optimizing blocking and washing conditions is crucial for specific TM9SF3 detection:

• Blocking: 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature is suitable for most applications. BSA-based blocking may be preferred when using phospho-specific antibodies in conjunction with TM9SF3 detection.

• Washing: Perform at least three 5-10 minute washes with TBST between each step (blocking, primary antibody, secondary antibody, and prior to detection).

• Background reduction: If high background is observed, increasing the washing time/frequency and reducing the antibody concentration can improve specificity.

• Antibody diluent: Prepare antibody dilutions in the same buffer used for blocking, typically with a reduced concentration (1-3% blocking agent).

What is the significance of TM9SF3 in gastric cancer progression?

TM9SF3 has been identified as a significant factor in gastric cancer (GC), particularly in the aggressive scirrhous-type:

• Expression patterns: Immunohistochemical analysis has demonstrated that approximately 50% (46 out of 91) of gastric cancer cases are positive for TM9SF3, with higher frequency observed in scirrhous-type GC .

• Clinical correlations: TM9SF3 expression shows significant correlation with:

  • Depth of tumor invasion

  • Advanced tumor stage

  • Undifferentiated GC

  • Poor patient outcomes (validated in multiple cohorts by both immunostaining and quantitative RT-PCR)

• Functional significance: Transient knockdown of TM9SF3 using siRNA resulted in decreased tumor cell-invasive capacity, suggesting a functional role in cancer cell invasion .

• Potential applications: Based on these findings, TM9SF3 is considered a potential diagnostic and therapeutic target for scirrhous-type gastric cancer .

How can researchers design experiments to investigate TM9SF3's role in tumor invasion?

To effectively investigate TM9SF3's role in tumor invasion, researchers should consider the following experimental approaches:

• Expression analysis in clinical samples:

  • Immunohistochemistry using validated TM9SF3 antibodies on tumor tissue microarrays with paired normal controls

  • Correlation of expression levels with clinicopathological parameters and patient outcomes

  • Quantitative RT-PCR validation of expression patterns

• In vitro functional studies:

  • siRNA or CRISPR-Cas9 mediated knockdown/knockout of TM9SF3 in appropriate cancer cell lines

  • Overexpression studies using TM9SF3 expression vectors

  • Assessing effects on:

    • Cell proliferation (MTT/WST-1 assays)

    • Migration (wound healing assays)

    • Invasion (Matrigel-coated Boyden chamber assays)

    • Colony formation (soft agar assays)

• Mechanistic studies:

  • Co-immunoprecipitation to identify TM9SF3 interaction partners

  • Subcellular localization studies using confocal microscopy

  • Analysis of downstream signaling pathways following TM9SF3 modulation

• In vivo models:

  • Xenograft models with TM9SF3-modulated cancer cells

  • Analysis of tumor growth, invasion, and metastasis

  • Correlation of TM9SF3 expression with markers of epithelial-mesenchymal transition

What controls should be included when using TM9SF3 antibodies in research applications?

Proper controls are essential for reliable TM9SF3 antibody-based experiments:

• Positive tissue controls: Mouse stomach tissue has been confirmed as a positive control for TM9SF3 antibody . For human samples, validated human tissues with known TM9SF3 expression should be included.

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at the same concentration)

  • Tissue known to have minimal TM9SF3 expression

• Knockdown/knockout validation: When available, samples from TM9SF3 knockdown or knockout experiments provide excellent specificity controls.

• Loading controls: For Western blot, appropriate loading controls (β-actin, GAPDH, etc.) should be included to normalize protein loading.

• Molecular weight markers: Always include molecular weight markers to confirm the detection of bands at the expected size (~68 kDa for TM9SF3, though observed MW may vary to 90 kDa in some systems) .

How can researchers validate antibody specificity for TM9SF3 detection?

To ensure antibody specificity for TM9SF3:

• Multiple antibody validation: Use at least two different antibodies targeting different epitopes of TM9SF3.

• Genetic manipulation: Compare antibody signal in wild-type versus TM9SF3 knockdown/knockout samples.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to block specific binding.

• Molecular weight confirmation: Confirm detection of appropriate molecular weight bands (~68 kDa calculated, although observed weight can vary) .

• Cross-reactivity testing: Test the antibody on samples from different species to confirm the specified reactivity (human, mouse) and lack of non-specific binding .

• Recombinant protein controls: Use purified recombinant TM9SF3 protein as a positive control when available.

What approaches can be used to study TM9SF3 interaction with other proteins?

To investigate TM9SF3's protein-protein interactions:

• Co-immunoprecipitation (Co-IP): Using TM9SF3 antibodies to pull down protein complexes, followed by mass spectrometry or Western blot for interacting partners.

• Proximity ligation assay (PLA): Allows visualization of protein interactions in situ with high sensitivity and specificity.

• FRET/BRET analysis: For studying dynamic protein interactions in living cells.

• Yeast two-hybrid screening: To identify novel interaction partners.

• GST pull-down assays: Using recombinant GST-tagged TM9SF3 fragments to identify direct binding partners.

• Crosslinking mass spectrometry: For mapping interaction interfaces with high resolution.

How can TM9SF3 be targeted for therapeutic development in cancer?

Based on research findings linking TM9SF3 to cancer progression , potential therapeutic strategies include:

• Antibody-based therapeutics:

  • Development of therapeutic antibodies targeting extracellular domains of TM9SF3

  • Antibody-drug conjugates for targeted delivery of cytotoxic agents

• RNA interference:

  • siRNA or shRNA delivery systems targeting TM9SF3 mRNA

  • Antisense oligonucleotides for TM9SF3 knockdown

• CRISPR-based approaches:

  • Gene editing to disrupt TM9SF3 function in cancer cells

• Small molecule inhibitors:

  • Development of inhibitors targeting TM9SF3 function or interactions

  • Structure-based drug design based on critical domains

• Biomarker applications:

  • Use of TM9SF3 as a diagnostic or prognostic marker in gastric cancer

  • Patient stratification for personalized treatment approaches