TMEM115 Antibody

What is TMEM115 and why is it important in cellular biology?

TMEM115 is an integral membrane protein enriched in the Golgi complex. It features four transmembrane domains in its N-terminal region, with both N- and C-terminal domains oriented toward the cytoplasm. This protein is evolutionarily conserved and plays a significant role in regulating Golgi-to-ER retrograde transport . It interacts with the conserved oligomeric Golgi (COG) complex and influences O-linked glycosylation, making it an important factor in secretory pathway research and potential disease mechanisms .

What are the common applications for TMEM115 antibodies in research?

TMEM115 antibodies are primarily used for:

• Western blotting (WB) to detect and quantify TMEM115 protein expression

• Immunofluorescence (IF) to visualize TMEM115 localization within the Golgi complex

• Immunohistochemistry (IHC) to examine TMEM115 expression in tissue sections

• ELISA for quantitative analysis of TMEM115 in samples

These applications enable researchers to study Golgi structure, trafficking pathways, and glycosylation processes in normal and pathological conditions.

How should I choose the appropriate TMEM115 antibody for my experiment?

When selecting a TMEM115 antibody, consider:

• Species reactivity: Ensure the antibody recognizes TMEM115 in your experimental model organism (human, mouse, etc.)

• Epitope location: For topology studies, select antibodies recognizing different domains. The C-terminal region (residues 255-344) is accessible in semi-permeabilized cells

• Validation data: Look for antibodies with validation in your specific application (WB, IF, IHC)

• Monoclonal vs. polyclonal: Monoclonals offer higher specificity; polyclonals provide stronger signals

The experimental goal should guide your choice. For subcellular localization studies, choose antibodies validated for immunofluorescence with demonstrated Golgi localization patterns .

What controls should I include when using TMEM115 antibodies?

Essential controls include:

• Positive control: Cell lines known to express TMEM115 (such as HEK293 cells with ~80,000 copies per cell)

• Negative control: TMEM115 knockdown cells using validated siRNA

• Secondary antibody-only control: To assess background staining

• Co-localization controls: Paired staining with established Golgi markers like GM130 (cis-Golgi), Mannosidase II (medial-Golgi), or GalT (trans-Golgi)

For functional studies, include BFA treatment (5 μg/ml) which causes redistribution of TMEM115 from the Golgi within 30-60 minutes, providing a specificity control .

How can I use TMEM115 antibodies to investigate Golgi topology and membrane organization?

To investigate Golgi topology using TMEM115 antibodies:

• Selective permeabilization technique: Use low concentrations of digitonin (5 μg/ml) to selectively permeabilize the plasma membrane while keeping the Golgi membrane intact. This allows assessment of whether the TMEM115 C-terminal epitope faces the cytoplasm

• Comparative analysis: Compare staining patterns with luminal Golgi proteins (such as GalT-GFP) and cytosolic Golgi proteins (such as GM130) under different permeabilization conditions

• Electron microscopy immunogold labeling: Use TMEM115 antibodies with gold-conjugated secondary antibodies to precisely localize TMEM115 within Golgi cisternae at ultrastructural level

This approach has confirmed that both N- and C-terminal domains of TMEM115 are oriented toward the cytoplasm, with the protein enriched in medial to trans Golgi cisternae .

How can TMEM115 antibodies be used in Golgi isolation and proteomics studies?

TMEM115 has become a valuable tool for Golgi isolation techniques:

• Golgi-IP methodology: Fuse TMEM115 to epitope tags (such as HA) to create GolgiTAG constructs for immunoprecipitation of intact Golgi mini-stacks

• Procedure overview:

  • Express tagged TMEM115 (GolgiTAG) in cells

  • Homogenize cells under conditions that preserve Golgi structure

  • Immunoprecipitate using anti-tag antibodies

  • Analyze isolated Golgi by proteomics, metabolomics, or lipidomics

• Advantages:

  • Minimal contamination from other organelles

  • Preserves functional Golgi mini-stacks

  • Enables multimodal analysis of Golgi content

This technique has successfully revealed the human Golgi proteome and metabolome, including the enrichment of UDP sugars involved in glycosylation processes .

Why might I observe non-specific labeling with TMEM115 antibodies in immunofluorescence studies?

Non-specific labeling with TMEM115 antibodies may result from:

• Fixation issues: TMEM115 is a membrane protein, so optimal fixation is critical

  • Recommendation: Test 4% paraformaldehyde (10 minutes) versus methanol fixation (-20°C, 5 minutes)

• Permeabilization problems: Excessive permeabilization may disrupt Golgi structure

  • Recommendation: Use mild detergents (0.1% Triton X-100 or 0.1% saponin) for whole-cell permeabilization

• Antibody concentration: Too high antibody concentration increases background

  • Recommendation: Perform titration experiments to determine optimal concentration

• Cross-reactivity: Antibodies may recognize similar epitopes in other proteins

  • Solution: Validate specificity using TMEM115 knockdown cells as negative controls

For optimal results, always include appropriate controls and co-labeling with established Golgi markers to confirm specific localization.

How can I resolve weak or absent signals when using TMEM115 antibodies in Western blotting?

To troubleshoot weak or absent TMEM115 signals in Western blotting:

• Sample preparation:

  • Add protease inhibitors to prevent degradation

  • Avoid boiling samples (use 37°C for 30 minutes instead), as membrane proteins can aggregate

  • Use appropriate lysis buffers containing 1% Triton X-100 or SDS for efficient solubilization

• Running conditions:

  • TMEM115 has a predicted molecular weight of ~40 kDa but may run differently due to post-translational modifications

  • Use gradient gels (4-20%) to better resolve membrane proteins

• Transfer optimization:

  • Use PVDF membranes (rather than nitrocellulose) for better retention of hydrophobic proteins

  • Add 0.1% SDS to transfer buffer to enhance elution of hydrophobic proteins from gel

• Detection enhancement:

  • Try extended primary antibody incubation (overnight at 4°C)

  • Use signal enhancers specific for membrane proteins

  • Consider more sensitive detection systems (such as ECL Prime or SuperSignal West Femto)

How can TMEM115 antibodies be used to study Golgi-to-ER retrograde transport?

TMEM115 antibodies enable sophisticated studies of retrograde transport:

• Brefeldin A (BFA) challenge assay:

  • Treat cells with BFA (5 μg/ml) for time course experiments (10-60 minutes)

  • Use TMEM115 antibodies to track redistribution patterns

  • Compare with other Golgi markers like GalT-GFP

  • Quantify the percentage of cells showing Golgi disassembly at each timepoint

• TMEM115 perturbation studies:

  • Knockdown TMEM115 using validated siRNA

  • Track delayed BFA-induced Golgi disassembly (~45-60 minutes compared to 30 minutes in control cells)

  • Analyze redistribution patterns of various Golgi residents

These approaches have revealed that proper TMEM115 expression levels are critical for efficient Golgi-to-ER retrograde transport, with both knockdown and overexpression causing delays in BFA-induced Golgi disassembly .

How can TMEM115 antibodies contribute to understanding glycosylation defects?

TMEM115 antibodies can help investigate glycosylation processes through:

• Combined glycosylation analysis:

  • Use TMEM115 antibodies to confirm knockdown or overexpression

  • Analyze O-linked glycosylation using lectins (PNA, HPA) that show reduced binding in TMEM115-depleted cells

  • Perform complementary biochemical assays of glycosyltransferase activities

• Co-immunoprecipitation studies:

  • Use TMEM115 antibodies to pull down protein complexes

  • Analyze interactions with COG complex components

  • Identify additional binding partners involved in glycosylation

• Rescue experiments:

  • Deplete endogenous TMEM115 and express siRNA-resistant mutants

  • Use TMEM115 antibodies to confirm expression of rescue constructs

  • Analyze restoration of glycosylation patterns and retrograde transport

These approaches connect TMEM115 function to broader Golgi homeostasis and glycosylation pathways critical in multiple diseases.

How should I quantify and interpret TMEM115 immunofluorescence data?

For robust quantification of TMEM115 immunofluorescence:

• Colocalization analysis:

  • Calculate Pearson's correlation coefficient between TMEM115 and Golgi markers

  • Expected values: Strong colocalization with manII-GFP (medial-Golgi) and partial overlap with GalT-GFP (trans-Golgi)

  • Limited colocalization with GM130 (cis-Golgi marker)

• Morphological quantification:

  • Measure Golgi area, compactness, and fragmentation index

  • TMEM115 knockdown typically results in more compact Golgi structure

• BFA response quantification:

  • Measure percentage of cells with redistributed Golgi markers over time

  • Control cells: ~80% redistribution by 30 minutes

  • TMEM115-silenced cells: ~30% redistribution by 30 minutes, ~70% by 60 minutes

Use standardized imaging parameters, blind analysis, and automated quantification tools to ensure reproducibility and minimize bias.

What are the important considerations when analyzing TMEM115 protein expression levels in different experimental conditions?

When analyzing TMEM115 protein expression:

• Reference standards:

  • Normal endogenous levels: ~80,000 copies per HEK293 cell

  • Include gradient of recombinant protein standards for absolute quantification

• Normalization approaches:

  • Normalize to total protein rather than single housekeeping genes

  • For cellular fractions, use compartment-specific markers (GM130 for Golgi)

  • Consider membrane protein-specific loading controls

• Expression comparison table:

• Interpretation guidelines:

  • Complete absence is rare even in knockdown conditions

  • Both under and overexpression can disrupt retrograde transport

  • Consider post-translational modifications that may affect antibody detection

How can TMEM115 antibodies be used to investigate Golgi dysfunction in disease models?

TMEM115 antibodies offer valuable insights into disease-related Golgi dysfunction:

• Neurodegenerative disease models:

  • Monitor TMEM115 localization in models of Parkinson's disease where Golgi fragmentation occurs

  • Track association with LRRK2 signaling pathways that dock at the Golgi

  • Analyze glycosylation defects in Alzheimer's and other neurodegenerative conditions

• Cancer cell studies:

  • Compare TMEM115 distribution in normal versus transformed cells

  • Investigate relationships between altered Golgi structure and cancer progression

  • Examine potential connections between TMEM115, retrograde transport, and altered glycosylation in cancer cells

• Methodology:

  • Combine TMEM115 antibody staining with super-resolution microscopy

  • Correlate structural changes with functional alterations in secretion and glycosylation

  • Use GolgiTAG-based approaches to isolate and analyze Golgi from disease models

The multi-functional nature of TMEM115 in Golgi structure, retrograde transport, and glycosylation makes it a valuable marker for studying Golgi pathobiology across diverse disease conditions.

What innovative research techniques combine TMEM115 antibodies with other methodologies?

Cutting-edge research approaches combining TMEM115 antibodies include:

• Golgi-IP proteomics/metabolomics:

  • Use TMEM115-based GolgiTAG to isolate intact Golgi

  • Perform LC-MS/MS analysis to characterize the Golgi proteome

  • Identify enriched metabolites, particularly UDP sugars involved in glycosylation

• Live-cell imaging strategies:

  • Correlate fixed-cell TMEM115 antibody staining with live-cell fluorescent protein tracking

  • Establish time-points for fixation and antibody staining based on live imaging

  • Develop nanobody-based tools derived from TMEM115 antibodies for live-cell applications

• Proximity labeling techniques:

  • Fuse TMEM115 to proximity labeling enzymes (BioID, APEX)

  • Map the spatial proteome surrounding TMEM115

  • Validate interactions using traditional TMEM115 antibody approaches

These innovative combinations of techniques provide multi-dimensional insights into Golgi biology beyond what can be achieved with single-methodology approaches.