TMEM40 Antibody

What is TMEM40 and what are its key biological characteristics?

TMEM40 is a 233 amino acid multi-pass membrane protein that exists in two isoforms and is localized to chromosome 3p25.1, a region known for involvement in various genetic disorders and cancers . This transmembrane protein plays significant roles in inflammatory processes, including collagen-induced arthritis . Its location within the cell membrane suggests involvement in cellular signaling pathways and interactions with extracellular matrix components, which are crucial for maintaining tissue integrity and mediating cellular responses to environmental cues . Chromosome 3, where TMEM40 is located, notably harbors a chemokine receptor gene cluster and several loci associated with human cancers, including key tumor suppressor genes that regulate apoptosis and cell migration .

What types of TMEM40 antibodies are currently available for research?

Research laboratories currently have access to several types of TMEM40 antibodies optimized for different experimental applications:

• Mouse monoclonal antibodies (e.g., TMEM40 Antibody A-9): These are available in both non-conjugated forms and conjugated variants including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates .

• Rabbit polyclonal antibodies: These provide alternative epitope recognition and are typically supplied at concentrations around 0.4 mg/ml .

Each antibody type offers distinct advantages depending on the experimental design, with monoclonals providing high specificity for particular epitopes and polyclonals offering broader detection capabilities across multiple epitopes.

What are the validated applications for TMEM40 antibodies?

TMEM40 antibodies have been validated for multiple experimental applications:

• Western blotting (WB): For protein expression quantification in tissue and cell lysates .

• Immunoprecipitation (IP): For isolating TMEM40 and associated protein complexes .

• Immunofluorescence (IF): For subcellular localization studies .

• Immunohistochemistry (IHC): For tissue expression analysis in clinical samples .

• Enzyme-linked immunosorbent assay (ELISA): For quantitative protein detection .

In validated studies, researchers have successfully employed these applications to investigate TMEM40's role in cancer progression, particularly in cutaneous squamous cell carcinoma (CSCC) and cervical cancer models .

How should tissue samples be prepared for TMEM40 immunohistochemistry?

For optimal TMEM40 detection in tissue samples, follow this validated protocol:

• Fix tissue samples with 4% paraformaldehyde for 12 hours at 4°C .

• Process and embed tissues in paraffin blocks .

• Prepare 6 μm thick sections for immunostaining .

• Deparaffinize sections in xylene and rehydrate using gradient ethanol solutions .

• Block sections in PBS containing 5% normal goat serum for 10 minutes at room temperature .

• Incubate with TMEM40-specific primary antibody (1:100 dilution) overnight at 4°C .

• Wash three times with 1X PBS .

• Incubate with appropriate HRP-conjugated secondary antibody (1:1,000 dilution) for 1 hour at room temperature .

• Visualize using a DAB Horseradish Peroxidase Color Development kit and counterstain with hematoxylin for 2 minutes at room temperature .

This protocol has been successfully employed in research examining TMEM40 expression in cancer tissues and control samples.

How can TMEM40 expression be accurately quantified in tissue microarrays?

For semi-quantitative assessment of TMEM40 expression in tissue microarrays, implement the following validated scoring system:

Percentage of positive cells scoring:

• Score 0: <5% positive cells

• Score 1: 5-25% positive cells

• Score 2: 26-50% positive cells

• Score 3: 51-75% positive cells

• Score 4: 76-100% positive cells

Staining intensity scoring:

• Score 0: No staining

• Score 1: Light yellow

• Score 2: Brown

• Score 3: Dark brown

Assessment should be performed by two independent observers using light microscopy at both 40× and 100× magnifications to ensure scoring reliability .

What are the optimal protocols for TMEM40 knockdown experiments?

For effective TMEM40 knockdown experiments, the following siRNA transfection protocol has been validated:

• Design target-specific siRNA sequences. A validated sequence for TMEM40 is 5′-GUGGACGCCUCUCAGUUAA-3′, with non-targeting negative control sequence 5′-TTCTCCGAACGTGTCACGT-3′ .

• Transfection procedure:

  • Use 5 nmol siRNA for transfection into cancer cells with Lipofectamine® 2000 according to manufacturer's instructions .

  • Incubate transfection mixture for 20 minutes at room temperature before adding to cells .

  • Use PBS treatment as control group .

• Transfection efficiency assessment:

  • Evaluate using flow cytometry 48 hours post-transfection .

  • If using fluorescently-labeled (e.g., FAM) siRNA, collect approximately 1×10^5 cells and analyze percentage of FAM-positive cells using flow cytometry .

  • Analyze data with FlowJo software (or equivalent) .

• Knockdown verification:

  • Confirm reduced TMEM40 expression by Western blot and RT-qPCR 48-72 hours post-transfection .

This protocol has been successfully employed in studies investigating TMEM40's role in cancer cell migration and invasion .

What RT-qPCR protocols are recommended for TMEM40 mRNA expression analysis?

For accurate quantification of TMEM40 mRNA expression, implement this validated RT-qPCR protocol:

• RNA extraction:

  • Extract total RNA from clinical samples or cell lines using a PureLink RNA Mini kit or equivalent .

  • Quantify RNA using a NanoDrop™ 2000 spectrophotometer .

• Reverse transcription:

  • Use a TaqMan MicroRNA Reverse Transcription kit to synthesize cDNA from total RNA .

• qPCR procedure:

  • Use SYBR-Green PCR kit on an ABI 7500 real-time PCR amplifier .

  • Thermocycling conditions: 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 34 seconds .

  • Use GAPDH as normalization control .

• Primer sequences:

• Data analysis:

  • Analyze relative gene expression data using the 2^(-ΔΔCq) method .

This protocol enables reliable quantification of TMEM40 expression changes in both clinical samples and experimental cell models.

How can TMEM40 antibodies be utilized to investigate cancer cell migration and invasion?

TMEM40 antibodies play a crucial role in validating cell migration and invasion assays after TMEM40 manipulation. The following protocols have been validated for studying TMEM40's influence on cancer cell behavior:

Wound healing assay protocol:

• Seed 1×10^4 cancer cells in 6-well plates and transfect with TMEM40-siRNA or overexpression vectors .

• Culture cells with serum-free medium to 85-90% confluence .

• Create wound gaps using a 10 μl RNase-free pipette tip .

• Image wound gaps at 0 and 48 hours using a light microscope (40× magnification) .

• Analyze migration by measuring the distance cells migrated in three different areas of each wound .

• Validate TMEM40 knockdown or overexpression by Western blot using appropriate TMEM40 antibodies (typically at 1:500 dilution) .

Transwell migration and invasion assay protocol:

• For invasion assays, pre-coat inserts with Matrigel for 4 hours at 37°C .

• Transfect cells with TMEM40-siRNA or overexpression vectors and incubate for 24 hours .

• Seed 3×10^4 cells in 300 μl of DMEM into the upper chamber of Transwell inserts (8-μm pore size) .

• Add 700 μl of DMEM supplemented with 20% FBS to the lower chambers .

• Incubate at 37°C in 5% CO₂ for 36 hours .

• Fix cells with 100% methanol for 5 minutes at room temperature and stain with 0.5% crystal violet for 15 minutes .

• Count migrating or invading cells in three randomly selected fields under a light microscope (40× magnification) .

• Calculate the average value using ImageJ software .

• Confirm TMEM40 expression changes using Western blot with appropriate TMEM40 antibodies .

These functional assays, coupled with TMEM40 antibody validation, provide robust methodologies for investigating TMEM40's role in cancer progression.

What is the optimal Western blot protocol for TMEM40 detection?

For optimal TMEM40 protein detection by Western blot, follow this validated protocol:

• Sample preparation:

  • Lyse tissue samples or cells and extract proteins using RIPA buffer containing 1 mM PMSF .

  • Determine protein concentrations using a BCA Protein Assay kit .

• Gel electrophoresis and transfer:

  • Separate approximately 40 μg of each total protein sample by SDS-PAGE on 10% gels .

  • Transfer proteins to nitrocellulose membranes .

• Blocking and antibody incubation:

  • Block membranes with 5% fat-free milk for 2 hours at room temperature .

  • Wash membranes with TBST .

  • Incubate overnight at 4°C with mouse anti-TMEM40 primary antibody (1:500 dilution; cat. no. sc-393601) .

  • Use mouse anti-GAPDH primary antibody (1:1,000 dilution) as loading control .

  • After washing, incubate membranes with appropriate HRP-conjugated secondary antibodies (1:5,000 dilution) for 1 hour at room temperature .

• Detection:

  • Detect signals using an enhanced chemiluminescent reagent kit .

  • For densitometric analysis, use ImageJ or similar software to quantify relative expression normalized to GAPDH .

This protocol has been successful in detecting TMEM40 expression differences between normal and cancer tissues, as well as in verifying knockdown or overexpression in experimental models .

How is TMEM40 expression associated with cancer progression?

Recent research has revealed contrasting roles for TMEM40 in different cancer types:

• In cutaneous squamous cell carcinoma (CSCC), reduced TMEM40 expression appears to inhibit cancer development, suggesting TMEM40 may promote CSCC progression .

• Conversely, in cervical cancer (CC), upregulation of TMEM40 is associated with malignant behavior, indicating TMEM40 may serve as an oncogenic driver in this cancer type .

These findings highlight the context-dependent nature of TMEM40's role in cancer biology. Researchers investigating TMEM40 should carefully consider the specific cancer type being studied and employ appropriate TMEM40 antibodies to accurately characterize expression patterns in their experimental systems.

What experimental designs are recommended for investigating TMEM40's role in cancer?

Based on published methodologies, a comprehensive investigation of TMEM40's role in cancer should include:

• Expression analysis:

  • Evaluate TMEM40 protein expression in paired normal and tumor tissues using Western blot and immunohistochemistry with validated antibodies .

  • Quantify TMEM40 mRNA levels using RT-qPCR in clinical samples and cell lines .

• Functional studies:

  • Perform TMEM40 knockdown using validated siRNA sequences .

  • Create TMEM40 overexpression models using full-length cDNA cloned into appropriate expression vectors .

  • Assess effects on cellular phenotypes including migration, invasion, proliferation, and apoptosis .

• Mechanism investigation:

  • Identify potential binding partners using co-immunoprecipitation with TMEM40 antibodies .

  • Evaluate effects on downstream signaling pathways using phospho-specific antibodies .

  • Consider chromosome 3p25.1 region interactions, particularly with chemokine receptor gene clusters and tumor suppressor genes .

This multi-faceted approach, centered around proper TMEM40 antibody utilization, provides a robust framework for elucidating TMEM40's function in cancer biology.