TMEM186 Antibody

What is TMEM186 and where is it localized in cells?

TMEM186 is a transmembrane protein associated with mitochondria. Research has demonstrated its localization through detergent permeabilization experiments that distinguish between different membrane compartments. Studies have examined TMEM186's relationship with nuclear membrane components, suggesting potential roles beyond simple mitochondrial functions . The protein shows different accessibility depending on whether only the plasma membrane (digitonin permeabilization) or all cellular membranes (TX100 permeabilization) are made permeable, indicating its specific membrane topology .

What applications are TMEM186 antibodies suitable for?

Commercial TMEM186 antibodies have been validated for several experimental applications:

These applications enable researchers to study TMEM186 expression, localization, and interactions in various experimental contexts .

What species reactivity do commercial TMEM186 antibodies exhibit?

Most commercially available TMEM186 antibodies show reactivity to human TMEM186, with some exhibiting cross-reactivity with mouse TMEM186. For instance, the polyclonal antibody targeting amino acids 124-213 exhibits reactivity to both human and mouse TMEM186, making it valuable for comparative studies across these species . When planning experiments involving different species, researchers should carefully verify the cross-reactivity information provided by manufacturers.

What are the available formats of TMEM186 antibodies?

TMEM186 antibodies are available in multiple formats to suit different experimental needs:

Each format offers distinct advantages depending on the experimental design and detection methods employed .

How should researchers optimize Western blotting protocols for TMEM186 detection?

For optimal Western blotting results with TMEM186 antibodies:

• Use recommended dilutions (1:500-1:5000) based on antibody sensitivity

• Consider specialized extraction protocols for membrane proteins

• Include positive controls (tissues/cells known to express TMEM186)

• Include negative controls (ideally TMEM186 knockout samples)

• Use protein G-purified antibodies (>95% purity) for highest specificity

• Be aware that different detergents may influence protein extraction efficiency

Researchers should note that TMEM186 is a transmembrane protein, which may require optimization of extraction and separation conditions different from those used for soluble proteins .

What considerations are important for immunofluorescence experiments with TMEM186 antibodies?

When performing immunofluorescence with TMEM186 antibodies:

• Begin with recommended dilutions (1:50-1:200) and optimize as needed

• Carefully select fixation methods that preserve mitochondrial structures

• Consider dual staining with established mitochondrial markers to confirm localization

• Both unconjugated antibodies with secondary detection or directly FITC-conjugated antibodies can be used

• When studying subcellular localization, differential permeabilization techniques can provide insights into protein topology

• For colocalization studies with nuclear structures, appropriate nuclear markers should be included

Research has utilized both FLAG-tagged TMEM186 constructs and TMEM186-specific antibodies to investigate its cellular localization and interactions .

How should TMEM186 antibodies be stored and handled to maintain optimal activity?

To maintain optimal antibody performance:

• Store concentrated antibody stock at -20°C or -80°C

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• Most commercial preparations contain 50% glycerol and 0.03% ProClin 300 as preservatives

• Note that ProClin 300 is classified as a hazardous substance requiring appropriate handling

• Working dilutions should be prepared fresh or stored at 4°C for short periods

• Follow manufacturer's recommendations regarding buffer compatibility

Proper storage is crucial as antibody degradation can lead to reduced sensitivity and increased background in experiments.

What controls should be included when using TMEM186 antibodies?

Robust experimental design requires appropriate controls:

• Positive controls: Tissues or cells known to express TMEM186 (based on transcript data)

• Negative controls:

  • Primary antibody omission

  • TMEM186 knockout samples (ideal negative control)

  • Tissues/cells with confirmed low TMEM186 expression

• Peptide competition controls: Pre-incubation with immunizing peptide should abolish specific signal

• For tagged constructs: Compare signals between tag-specific and TMEM186-specific antibodies

Research has utilized TMEM186 knockout cell lines as important negative controls to validate antibody specificity and to study the effects of TMEM186 deletion .

How can TMEM186 antibodies be employed to study protein-protein interactions?

TMEM186 antibodies can facilitate several approaches to study protein interactions:

• Immunoprecipitation (IP) followed by mass spectrometry

• Co-immunoprecipitation (Co-IP) to confirm specific interactions

• Proximity ligation assays for in situ detection of protein interactions

• Biomolecular fluorescence complementation (BiFC) assays

Research has successfully employed FLAG-tagged TMEM186 with immunoprecipitation and mass spectrometry to identify interactions with nuclear pore complex proteins including NDC1, AAAS, and NUP35/53. These interactions were subsequently confirmed using BiFC assays .

How can researchers validate TMEM186 knockout models using antibodies?

When validating knockout models:

• Compare antibody signal between wild-type and knockout samples across multiple techniques

• Use quantitative approaches (Western blot densitometry) to detect any residual expression

• Be aware that some knockout models may retain minimal expression (e.g., mouse models showed 2.1% residual TMEM186 transcript)

• Complement protein detection with transcript analysis (qRT-PCR)

• Consider using multiple antibodies targeting different epitopes

Research has successfully employed TMEM186 antibodies to validate knockout cell lines, analyzing the resulting effects on mitochondrial complex I using blue native PAGE (BN-PAGE) .

What approaches can help distinguish between specific and non-specific antibody binding?

To ensure signal specificity:

• Titrate antibody concentrations to determine optimal signal-to-noise ratio

• Compare staining patterns across multiple antibodies targeting different epitopes

• Use siRNA knockdown to verify signal reduction correlates with reduced expression

• Perform peptide competition assays

• Analyze signal in tissues with known expression patterns based on transcriptomic data

• When possible, include genetic models (knockout, knockdown) as gold-standard controls

Researchers have used multiple validation approaches, including genetic models, to ensure specificity when studying TMEM186 localization and function .

How can TMEM186 antibodies be used to study protein localization at the subcellular level?

For detailed subcellular localization studies:

• Use differential detergent permeabilization:

  • Digitonin (40 μg/mL) selectively permeabilizes the plasma membrane

  • TX100 permeabilizes all cellular membranes including nuclear membrane

• Compare with known compartment markers:

  • Lamin B (nuclear membrane, internal face)

  • Calnexin (spans nuclear membrane with C-terminus in cytoplasm)

• Use super-resolution microscopy for precise localization

• Consider dual-labeling with organelle-specific markers

Research has employed these approaches to determine TMEM186's membrane topology and relationships with nuclear structures .

What is known about TMEM186's role in mitochondrial function?

TMEM186 has been implicated in mitochondrial function:

• It co-migrates with components of the Mitochondrial Complex I Assembly (MCIA) complex based on dynamic complexome profiling

• TMEM186 was significantly enriched in NDUFAF1-BirA* proximity labeling experiments

• Analysis of TMEM186 knockout cell lines by BN-PAGE showed impacts on complex I assembly

• The protein is classified with "mitochondrion" as its background in protein databases

These findings suggest TMEM186 may play an important role in mitochondrial complex I assembly or function, though the precise mechanisms require further investigation .

How is TMEM186 implicated in obesity and appetite control?

Research has revealed connections between TMEM186 and metabolism regulation:

Studies showed that TMEM186 expression in the paraventricular nucleus (PVN) of the hypothalamus is altered by nutritional state. Overexpression of TMEM186 using adeno-associated viral vectors in the PVN resulted in significantly reduced weight gain compared to controls (0.9g vs 2.7g after 6 weeks), suggesting a role in appetite control and body weight regulation .

What experimental models are available for studying TMEM186 function?

Several experimental models have been developed:

• Knockout mice: Generated through targeting disruption of exon 2 of TMEM186, resulting in only 2.1% residual expression

• Heterozygous mouse models: Show approximately 50% reduction in TMEM186 expression

• Cell line knockout models: Generated for studying effects on mitochondrial function

• Overexpression models: Using adeno-associated viral vectors (AAV-TMEM186) for targeted expression

• Tagged expression systems: FLAG-tagged TMEM186 constructs for protein interaction studies

These models provide complementary approaches to investigate TMEM186 function in different contexts and at different levels of biological organization .

How do TMEM186 interactions with nuclear pore complex proteins inform its function?

The identification of interactions between TMEM186 and nuclear pore complex proteins raises interesting functional possibilities:

• Mass spectrometry identified interactions with three nuclear pore components (NDC1, AAAS, NUP35/53)

• These proteins showed high abundance in pulldown experiments, suggesting robust interactions

• BiFC assays confirmed these protein-protein interactions

• These findings suggest potential roles beyond classical mitochondrial functions

• TMEM186 may participate in communication between mitochondria and the nucleus

• It may have roles in nuclear-mitochondrial protein trafficking

The dual association with both mitochondrial complexes and nuclear pore components suggests TMEM186 may function at the interface of these critical cellular structures .

What buffer systems are optimal for TMEM186 antibody applications?

When working with TMEM186 antibodies, buffer selection can significantly impact results:

• Commercial antibodies are typically supplied in:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% ProClin 300 as preservative

• For Western blotting, standard transfer buffers are generally suitable

• For immunoprecipitation, consider mild non-ionic detergents that preserve protein interactions

• For immunohistochemistry, optimize antigen retrieval buffers (citrate, EDTA, or Tris-based)

• For ELISA applications, standard blocking buffers (BSA or casein-based) are appropriate

Proper buffer selection helps maintain antibody stability and specificity while maximizing target antigen accessibility.

How can researchers troubleshoot inconsistent results with TMEM186 antibodies?

When facing inconsistent results:

• Verify antibody lot consistency (request Certificate of Analysis from manufacturer)

• Check protein extraction efficiency for membrane proteins

• Optimize detergent concentration for solubilization without disrupting epitopes

• Consider native versus denaturing conditions (especially important for transmembrane proteins)

• Evaluate fixation methods that may affect epitope accessibility

• Test multiple antibodies against different epitopes of TMEM186

• Consider post-translational modifications that might affect antibody recognition

Systematic troubleshooting approaches can help distinguish between technical issues and biologically relevant variations in TMEM186 detection.

What techniques can researchers use to study TMEM186 in complex tissue samples?

For complex tissue analysis:

• Immunohistochemistry with optimized antigen retrieval protocols

• Laser capture microdissection combined with Western blotting for region-specific analysis

• In situ hybridization to correlate protein with mRNA localization

• Multi-label immunofluorescence to examine co-localization with cell-type specific markers

• Tissue clearing techniques combined with immunofluorescence for 3D visualization

• Single-cell approaches to examine cell-type specific expression patterns

Research has successfully employed various approaches to study TMEM186 expression and function in brain regions like the paraventricular nucleus of the hypothalamus .

How can researchers quantitatively assess TMEM186 expression levels?

For quantitative TMEM186 expression analysis:

• Western blotting with densitometry (normalize to appropriate loading controls)

• Quantitative immunofluorescence (measure fluorescence intensity)

• ELISA-based quantification (using validated TMEM186 antibodies)

• Correlate protein levels with transcript quantification (qRT-PCR)

• Consider challenges of membrane protein quantification:

  • Extraction efficiency may vary

  • Epitope accessibility may be affected by protein conformation

  • Post-translational modifications may influence antibody binding

When examining expression changes, it's important to use multiple complementary approaches, as seen in studies of TMEM186 knockout models where both protein and transcript levels were assessed .