TMEM127 Antibody, HRP conjugated

What is TMEM127 and what cellular compartments does it localize to?

TMEM127 is a transmembrane protein encoded by the TMEM127 tumor suppressor gene, functioning primarily at endosomal compartments. Wild-type TMEM127 displays a characteristic punctate localization pattern, with approximately 68% of TMEM127-positive structures colocalizing with the early endosomal marker Rab5 and about 38% with the Rab5 effector EEA1 . This protein also demonstrates dynamic localization to lysosomes, with increased association following amino acid stimulation, mirroring the pattern observed with mTORC1 .

Unlike wild-type TMEM127, certain mutant forms (such as S147del found in renal cell carcinoma) show diffuse cytoplasmic distribution, indicating disrupted subcellular targeting . These localization differences are functionally significant, as proper endosomal association is required for TMEM127's tumor suppressor activities.

What are the optimal conditions for TMEM127 antibody detection in western blot applications?

For optimal western blot detection of TMEM127 using HRP-conjugated antibodies:

Sample preparation:

• Use RIPA or NP-40 based lysis buffers containing protease inhibitors to prevent degradation

• Include membrane fractionation steps when analyzing subcellular distribution

• Process samples at 4°C to minimize protein degradation

Electrophoresis and transfer parameters:

• TMEM127 has a molecular weight of approximately 26 kDa; use 10-15% polyacrylamide gels

• For transmembrane proteins like TMEM127, wet transfer with methanol-containing buffers improves transfer efficiency

• Optimal transfer conditions: 100V for 1-2 hours or 30V overnight at 4°C

Antibody incubation:

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

• For HRP-conjugated antibodies, dilution ranges of 1:1000-1:5000 are typically effective

• Extended incubation (overnight at 4°C) often provides cleaner signals

TMEM127 detection can be challenging due to variable expression levels across tissues. Multiple positive controls should be included, such as TMEM127-overexpressing cells alongside endogenously expressing cell lines like SH-SY5Y neuroblastoma cells .

How can I validate the specificity of TMEM127 antibodies in my experimental system?

Comprehensive validation of TMEM127 antibodies requires multiple complementary approaches:

Genetic controls:

• Compare detection in wild-type versus TMEM127 knockout cells (CRISPR-Cas9 generated)

• Use siRNA knockdown to confirm signal reduction corresponds with decreased protein levels

• Test in TMEM127 null mouse embryonic fibroblasts (MEFs)

Expression controls:

• Overexpress tagged TMEM127 constructs as positive controls

• Compare signal with antibodies targeting different TMEM127 epitopes

• Test reactivity with recombinant TMEM127 protein of known concentration

Application-specific validation:

• For immunofluorescence: verify punctate endosomal pattern in wild-type cells versus diffuse distribution in cells expressing mutant TMEM127 (e.g., S147del)

• For immunoprecipitation: confirm enrichment of TMEM127 interaction partners like Rab5

• For western blot: verify single band at expected molecular weight that disappears in knockout models

Signal validation is particularly important when studying TMEM127 mutations, as some mutations affect protein stability while others primarily disrupt localization without altering expression levels .

How should I detect TMEM127 colocalization with endosomal markers?

Detecting TMEM127 colocalization with endosomal markers requires optimized immunofluorescence protocols:

Sample preparation:

• Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

• Gentle permeabilization (0.1% Triton X-100, 5 minutes) preserves membrane structures

• Block with 3% BSA in PBS to reduce non-specific binding

Antibody selection and optimization:

• Use validated antibodies against endosomal markers: Rab5 (early endosomes), EEA1 (early endosome effector), LAMP2 (lysosomes)

• Optimize antibody concentrations to achieve comparable signal intensities

• Select secondary antibodies with minimal spectral overlap

Imaging parameters:

• Use confocal microscopy with appropriate optical sectioning

• Acquire z-stacks (0.3-0.5 μm step size) to capture complete volume

• Apply deconvolution to improve signal-to-noise ratio

Quantification methods:

• Calculate Pearson's or Mander's correlation coefficients

• Perform object-based colocalization analysis

• Measure percent of TMEM127 puncta positive for each marker

Previous studies have established that approximately 68±4.2% of wild-type TMEM127-positive structures colocalize with Rab5 and 38±1.9% with EEA1 . These benchmarks provide useful comparison points when analyzing mutant forms or experimental manipulations.

What controls should I include when studying TMEM127 mutations?

When studying TMEM127 mutations, include these essential controls:

Positive controls:

• Wild-type TMEM127 expression construct

• Endogenous TMEM127 in appropriate cell lines (HEK293T, HeLa, SH-SY5Y)

• Known functional readouts (e.g., normal endosomal localization, Rab5 interaction)

Negative controls:

• Empty vector transfection

• TMEM127 knockout cells

• Irrelevant proteins with similar molecular weight or localization patterns

Mutation-specific controls:

• Previously characterized TMEM127 mutations (S147del, D70N, P118L, T126I)

• Multiple independent mutations affecting the same functional domain

• Silent mutations that preserve amino acid sequence

Functional validation:

• Endosomal localization assays (colocalization with Rab5/EEA1)

• Rab5Q79L-mediated endosomal fusion assessment

• mTOR signaling pathway activation (phosphorylation of S6K, S6, 4EBP1)

• RET membrane accumulation measurements

Research shows that TMEM127 mutations have varied effects: S147del causes complete mislocalization, D70N shows partial mislocalization, while P118L and T126I maintain punctate distribution similar to wild-type TMEM127 . These differential phenotypes provide valuable benchmarks for characterizing novel mutations.

How can I investigate TMEM127's role in regulating mTOR signaling using TMEM127 antibodies?

To investigate TMEM127's role in mTOR regulation:

Signaling pathway analysis:

• Assess phosphorylation of direct mTOR targets (S6K, S6, 4EBP1) by western blotting

• Compare basal vs. amino acid-stimulated signaling in TMEM127 wild-type vs. knockout cells

• Examine whether TMEM127 mutants can rescue normal mTOR regulation

Dynamic recruitment studies:

• Perform time-course analysis of TMEM127 and mTOR recruitment to lysosomes after amino acid stimulation

• Quantify TMEM127-mTOR colocalization (peaks approximately 10 minutes post-stimulation)

• Track TMEM127-LAMP2 association (lysosomes) following amino acid exposure

Interaction analysis:

• Immunoprecipitate TMEM127 to identify associated mTOR pathway components

• Analyze TMEM127 associations in nutrient-rich vs. starved conditions

• Compare interactions in wild-type vs. cancer-associated mutants

Research demonstrates that TMEM127 reduces phosphorylation of mTOR targets and shows dynamic localization to lysosomes following amino acid stimulation . TMEM127 appears to cooperate with activated Rab5 to suppress mTOR signaling, as co-expression of wild-type TMEM127 with constitutively active Rab5Q79L enhances this inhibitory effect . Notably, RCC-associated TMEM127 mutants show impaired ability to suppress mTOR signaling, providing a mechanistic link to their oncogenic potential .

What methodologies can I use to study TMEM127's interaction with Rab5 and early endosomes?

To study TMEM127-Rab5 interactions and endosomal functions:

Co-immunoprecipitation approaches:

• Use anti-TMEM127 antibodies to immunoprecipitate protein complexes followed by Rab5 detection

• Perform reciprocal immunoprecipitation with Rab5 antibodies

• Compare interaction efficiency with different Rab5 mutants:

  • Rab5WT (wild-type)

  • Rab5Q79L (constitutively active, GTP-bound)

  • Rab5S34N (inactive, GDP-bound)

Advanced microscopy techniques:

• Implement super-resolution microscopy for detailed colocalization analysis

• Use quantitative colocalization metrics to assess overlap with early endosomal markers

• Track dynamic interactions through live-cell imaging

Functional endosome assays:

• Quantify enlarged Rab5Q79L-positive vesicles in TMEM127-expressing vs. deficient cells

• Measure early endosome formation using EEA1 puncta quantification

• Assess endosome maturation rates through Rab5-to-Rab7 conversion kinetics

Research demonstrates that TMEM127 associates with Rab5-containing protein complexes in a GTP-dependent manner, with increased association in cells expressing GTP-bound Rab5 . TMEM127-null cells show striking decreases in enlarged Rab5Q79L-positive vesicles, indicating that TMEM127 is required for Rab5-mediated endosomal fusion . This phenotype can be rescued by wild-type TMEM127 expression but not by the TMEM127-S147del mutant , providing a functional readout for mutant analysis.

How can I distinguish between wild-type and mutant TMEM127 proteins in experimental systems?

Distinguishing wild-type from mutant TMEM127 requires multi-faceted approaches:

Localization analysis:

• Implement high-resolution confocal microscopy to visualize distribution patterns

• Quantify punctate vs. diffuse cytoplasmic localization

• Measure colocalization coefficients with organelle markers:

  • Wild-type: 68±4.2% colocalization with Rab5, 38±1.9% with EEA1

  • S147del mutant: Diffuse cytoplasmic pattern with minimal organelle colocalization

  • D70N mutant: Diffuse in ~33% of cells

  • P118L and T126I mutants: Predominantly punctate, similar to wild-type

Functional assessments:

• Evaluate rescue capacity in TMEM127-null backgrounds

• Assess effects on:

  • Rab5-mediated endosomal fusion (enlarged vesicle formation with Rab5Q79L)

  • Early endosome formation (EEA1 puncta quantification)

  • mTOR signaling (target phosphorylation of S6K, S6, 4EBP1)

  • RET protein accumulation at cell surface

Biochemical approaches:

• Compare protein stability through cycloheximide chase experiments

• Analyze protein-protein interactions via co-immunoprecipitation with known partners

• Assess post-translational modifications that might differ between variants

Research shows that TMEM127 mutations have variable effects on protein function: S147del causes complete cytoplasmic mislocalization and functional loss, D70N shows intermediate phenotypes, while P118L and T126I retain substantial wild-type characteristics despite being cancer-associated , suggesting potentially more subtle functional defects in these variants.

How can I investigate TMEM127's role in RET protein regulation and signaling?

To study TMEM127's impact on RET regulation:

Surface protein analysis:

• Implement surface biotinylation assays to specifically quantify membrane-localized RET

• Compare surface/total RET ratios in TMEM127 wild-type vs. knockout cells

• Rescue experiments should show approximately 5-fold reduction in surface RET when wild-type TMEM127 is reintroduced

RET trafficking dynamics:

• Study RET internalization kinetics using antibody-feeding assays

• Measure RET protein half-life through cycloheximide chase experiments

• Track RET trafficking through endosomal compartments via immunofluorescence

Signaling activation assessment:

• Quantify phosphorylation of RET and downstream effectors (MAPK, PI3K/AKT)

• Compare constitutive vs. ligand-induced activation in TMEM127-deficient cells

• Test RET inhibitor sensitivity in TMEM127 wild-type vs. knockout backgrounds

Transcriptional feedback analysis:

• Measure RET mRNA levels in TMEM127-manipulated cells

• Analyze potential feedback mechanisms (TMEM127-KO cells show decreased RET mRNA despite increased protein levels)

• Investigate transcription factor activity at the RET promoter

Research demonstrates that TMEM127 loss leads to RET protein accumulation at the cell surface and constitutive activation . This appears to result from impaired membrane dynamics and reduced efficiency of clathrin-mediated endocytosis rather than transcriptional upregulation, as TMEM127-KO cells actually show decreased RET mRNA levels . Surface biotinylation assays revealed approximately 5-fold increases in cell surface RET in TMEM127-knockout compared to control cells .

What techniques can I use to study TMEM127's effects on membrane dynamics and protein trafficking?

To investigate TMEM127's role in membrane dynamics and trafficking:

Membrane protein mobility measurements:

• Implement fluorescence recovery after photobleaching (FRAP) to quantify diffusion rates

• Use single-particle tracking to analyze movement dynamics at the molecular level

• Apply fluorescence correlation spectroscopy (FCS) to measure protein diffusion in live membranes

Endocytosis and trafficking assays:

• Quantify uptake rates of endocytic cargo proteins (transferrin, EGF, etc.)

• Measure internalization kinetics of membrane proteins in TMEM127 wild-type vs. knockout cells

• Track vesicle progression through the endolysosomal system using pulse-chase approaches

Membrane domain analysis:

• Assess lipid raft composition and protein distribution using detergent resistance assays

• Implement super-resolution microscopy to visualize nanoscale membrane organization

• Analyze membrane fluidity using environment-sensitive fluorescent probes

Global trafficking assessment:

• Examine multiple transmembrane proteins (beyond RET) to determine specificity

• Other proteins affected by TMEM127 loss include N-cadherin and transferrin receptor-1

• Investigate clathrin-coated pit formation and maturation kinetics

Research indicates that TMEM127 loss increases membrane protein diffusability and impairs normal membrane transitions and protein complex stabilization . This leads to inappropriate accumulation of signaling receptors like RET at the cell surface, resulting in constitutive activation . The effects appear to be relatively global rather than specific to individual proteins, suggesting that TMEM127 plays a fundamental role in membrane organization and dynamics that impacts multiple trafficking pathways .