TMEM127 Antibody, FITC conjugated

What is the optimal application range for TMEM127 antibody, FITC conjugated?

TMEM127 antibody conjugated with FITC is primarily used for fluorescence-based applications including immunofluorescence microscopy and flow cytometry. The antibody specifically targets the amino acid region 1-95 of the human TMEM127 protein, allowing for direct visualization of TMEM127 localization without secondary antibody requirements . For optimal results in immunofluorescence applications, researchers should begin with dilutions in the range of 1:200-1:800, then optimize based on specific experimental conditions and cell types .

How should samples be prepared for TMEM127 localization studies using FITC-conjugated antibodies?

For effective TMEM127 localization studies:

• Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Block with 1-5% BSA in PBS for 30-60 minutes

• Incubate with TMEM127-FITC antibody at appropriate dilution (starting at 1:200-1:800)

• Wash thoroughly with PBS (3-5 times)

• Mount with anti-fade mounting medium containing DAPI for nuclear counterstaining

This protocol is particularly effective for visualizing TMEM127's endosomal localization patterns, which partially overlap with early endosomal markers like Rab5 and EEA1 .

What controls should be included when using TMEM127 antibody, FITC conjugated?

When utilizing FITC-conjugated TMEM127 antibodies, the following controls are essential:

These controls are critical for interpreting subcellular localization data, particularly when examining TMEM127's dynamic association with endosomal and lysosomal compartments .

How can FITC-conjugated TMEM127 antibodies be used to track dynamic protein-protein interactions in live cells?

For tracking dynamic TMEM127 interactions in live cells:

• Optimize antibody loading using protein transfection reagents (e.g., Chariot™ or BioPORTER®) at concentrations of 0.5-2 μg antibody per well

• Maintain physiological conditions (37°C, 5% CO₂) during imaging

• Utilize rapid acquisition confocal microscopy with sensitive detectors to minimize photobleaching

• For amino acid stimulation experiments, starve cells for 50-60 minutes and then add amino acid mixtures while imaging

• Quantify colocalization with other fluorescently tagged proteins using Pearson's coefficient or Manders' overlap coefficient

This approach can be particularly valuable for studying the dynamic association between TMEM127 and the lysosomal marker LAMP2, which increases after amino acid exposure, peaking at approximately 10 minutes before gradually decreasing . Similar dynamics are observed with mTOR colocalization, suggesting coordinated recruitment to lysosomes upon nutrient stimulation.

What are the methodological considerations when studying mutated TMEM127 variants using immunofluorescence?

When investigating mutated TMEM127 variants:

• Generate expression constructs for wild-type and mutant TMEM127 (e.g., S147del, D70N, P118L, T126I mutations identified in renal cell carcinomas)

• Transfect into appropriate cell models with low endogenous TMEM127 expression

• Use FITC-conjugated TMEM127 antibodies alongside organelle markers for colocalization studies

• Quantify the degree of overlap between TMEM127 variants and early endosomal markers (Rab5, EEA1)

• Compare subcellular distribution patterns between wild-type and mutant proteins

Research has demonstrated that certain mutations (S147del, D70N) significantly reduce TMEM127's association with early endosomal domains, potentially explaining their pathogenic effects . The precise quantification of colocalization revealed that wild-type TMEM127 shows 68±4.2% colocalization with Rab5 and 38±1.9% with EEA1, values that are significantly reduced in certain mutants .

How can TMEM127-FITC antibodies be used to investigate the relationship between TMEM127 and mTORC1 signaling?

To study TMEM127's role in mTORC1 signaling:

• Design dual-labeling experiments using TMEM127-FITC antibody and antibodies against mTORC1 components (mTOR, Raptor) or downstream targets (phospho-S6K, phospho-4EBP1)

• Perform amino acid starvation and re-stimulation protocols:

  • Starve cells in EBSS for 50-60 minutes

  • Stimulate with complete amino acid mixture for various timepoints (5-60 minutes)

• Analyze colocalization patterns and intensities at lysosomal regions (marked by LAMP2)

• Compare wild-type cells with TMEM127 knockout/knockdown models

• Quantify phosphorylation of mTORC1 targets by immunoblotting in parallel experiments

Research indicates that TMEM127 is recruited to lysosomes upon amino acid stimulation with dynamics similar to mTORC1, suggesting coordinated regulation . Cells lacking TMEM127 show increased lysosomal accumulation of LAMTOR proteins, which may enhance mTORC1 recruitment and activation .

What technical challenges exist when using FITC-conjugated antibodies for studying TMEM127 in tissue samples, and how can they be overcome?

Challenges in tissue immunofluorescence with FITC-conjugated TMEM127 antibodies include:

Researchers studying TMEM127 in tissues should note that antigen retrieval with TE buffer pH 9.0 has been specifically recommended for optimal results, though citrate buffer pH 6.0 may serve as an alternative .

How should experiments be designed to investigate TMEM127's dynamic association with different endosomal compartments?

For comprehensive endosomal trafficking studies:

• Design a time-course experiment with FITC-TMEM127 antibody alongside markers for:

  • Early endosomes (Rab5, EEA1)

  • Late endosomes (Rab7)

  • Lysosomes (LAMP2)

  • Recycling endosomes (Rab11)

• Implement quantitative colocalization analysis:

  • Calculate Pearson's correlation coefficient

  • Determine percent overlap

  • Measure intensity correlation quotients

• Manipulate endosomal dynamics using:

  • Constitutively active Rab5 (Rab5Q79L) to enlarge early endosomes

  • Dominant-negative Rab5 (Rab5S34N) to inhibit early endosomal fusion

  • Bafilomycin A1 to inhibit lysosomal acidification

Studies have shown that TMEM127 colocalizes with early endosomes in a Rab5-dependent manner, with constitutively active Rab5Q79L increasing TMEM127 association with enlarged vesicles, while dominant-negative Rab5S34N causes dispersion of both Rab5 and TMEM127 signals .

How can researchers accurately assess the functional impact of TMEM127 mutations on mTORC1 signaling?

To comprehensively evaluate TMEM127 mutation effects:

• Generate cell models expressing wild-type or mutant TMEM127 in a TMEM127-null background

• Assess mTORC1 signaling through multiple complementary approaches:

  • Immunofluorescence to track mTORC1 lysosomal localization

  • Western blotting to quantify phosphorylation of multiple mTORC1 targets (S6K, S6, 4EBP1)

  • Proximity ligation assays to detect protein-protein interactions

  • Live-cell imaging to monitor dynamic recruitment kinetics

• Conduct nutrient response experiments:

  • Amino acid starvation/re-stimulation

  • Growth factor withdrawal/re-addition

  • Combined nutrient manipulations

• Compare results across multiple cell types to ensure generalizability

Research has demonstrated that certain TMEM127 mutations fail to reduce mTOR target phosphorylation when co-expressed with activated Rab5, unlike wild-type TMEM127 which cooperates with Rab5 to inhibit mTORC1 signaling .

How should researchers resolve conflicting data regarding TMEM127 subcellular localization?

When facing contradictory TMEM127 localization data:

• Systematically evaluate experimental variables:

  • Antibody epitopes (N-terminal AA 1-95 vs. other regions)

  • Fixation methods (paraformaldehyde vs. methanol)

  • Cell types (endogenous expression levels vary)

  • Expression systems (overexpression may alter localization)

• Employ multiple detection methods:

  • Subcellular fractionation with immunoblotting

  • Immunofluorescence with super-resolution microscopy

  • Electron microscopy with immunogold labeling

  • Live-cell imaging with fluorescent protein fusions

• Quantify overlap with multiple organelle markers:

  • Early endosomes: Rab5, EEA1 (68±4.2% and 38±1.9% colocalization respectively)

  • Late endosomes: Rab7

  • Lysosomes: LAMP2 (dynamic association following amino acid stimulation)

  • Golgi apparatus: GM130

  • Endoplasmic reticulum: Calnexin

Research has shown that TMEM127 localizes dynamically to multiple membrane compartments, with the strongest association observed with early endosomes and lysosomes in a nutrient-dependent manner .

What are the best practices for analyzing TMEM127's role in endosomal fusion events?

For rigorous analysis of TMEM127's role in endosomal dynamics:

• Quantify endosomal parameters in TMEM127 wild-type versus knockout/knockdown cells:

  • Number of EEA1-positive puncta per cell

  • Size distribution of endosomal vesicles

  • Rate of endosomal fusion events in live cells

  • Trafficking of endocytosed cargo

• Implement fusion assays:

  • Fluorescent dextran pulse-chase experiments

  • Dual-color early endosome fusion assays

  • EGF receptor degradation kinetics

• Rescue experiments with:

  • Wild-type TMEM127

  • Domain-specific mutants

  • siRNA-resistant constructs

Studies have demonstrated that TMEM127 deficiency results in decreased early endosomal (EEA1-positive) puncta and increased late endosomal/lysosomal (Rab7/LAMP2-positive) vesicles, suggesting a role in endosomal maturation and fusion events .

How can TMEM127-FITC antibodies be employed in high-content screening approaches?

For high-content screening with TMEM127-FITC antibodies:

• Establish automated imaging platforms:

  • 96/384-well format compatible with high-content microscopy

  • Multi-parameter phenotypic readouts

  • Machine learning-based image analysis

• Design TMEM127-focused screens:

  • Small molecule libraries targeting endosomal trafficking

  • siRNA/CRISPR libraries for genetic interaction partners

  • Compound libraries affecting mTORC1 signaling

• Develop quantitative phenotypic profiles:

  • TMEM127 localization patterns

  • Endosomal morphology metrics

  • mTORC1 activation status

  • Co-localization with lysosomal markers

• Validate hits with secondary assays:

  • Biochemical interaction studies

  • Functional readouts of mTORC1 activity

  • Endosomal trafficking kinetics

This approach could identify novel regulators of TMEM127 function or compounds that modulate its tumor suppressor activity through effects on endosomal trafficking and mTORC1 signaling.

What methodological considerations are important when studying TMEM127 in patient-derived samples?

For translational studies with patient materials:

• Tissue preparation considerations:

  • Rapid fixation to preserve antigenicity

  • Optimized antigen retrieval (TE buffer pH 9.0 recommended)

  • Autofluorescence quenching for FITC detection

• Analytical approaches:

  • Multiplex immunofluorescence to correlate TMEM127 with disease markers

  • Digital pathology with quantitative image analysis

  • Tissue microarrays for high-throughput analysis

• Integration with molecular profiling:

  • Correlation with TMEM127 mutation status

  • Association with mTORC1 pathway activation

  • Co-analysis with endosomal trafficking markers

• Validation strategies:

  • Multiple antibodies targeting different TMEM127 epitopes

  • RNA-scope for mRNA expression correlation

  • Patient-derived cell models for functional studies

Researchers should note that TMEM127 antibodies have been validated for immunohistochemistry applications in multiple human tissues including stomach cancer, heart, and liver cancer tissues .