TMEM26 Antibody

What is TMEM26 and why is it significant for research?

TMEM26 is a multi-pass transmembrane protein with a canonical length of 368 amino acid residues and a molecular mass of approximately 41.7 kDa in humans. Its primary subcellular localization is in the plasma membrane, making it an interesting target for cell surface studies . TMEM26 has gained significance in cancer research, particularly in Esophageal Squamous Cell Carcinoma (ESCC), where it appears to play a critical role in epithelial-mesenchymal transition (EMT) by disrupting tight junction formation and promoting NF-κB signaling . Additionally, TMEM26 shows specific expression in retinal tissue, particularly in the Inner Plexiform Layer (IPL) and Outer Plexiform Layer (OPL), with a potential weak association with Primary Open-Angle Glaucoma (POAG) .

What different forms of TMEM26 protein exist and how do antibodies detect them?

Research has identified multiple TMEM26 protein isoforms with different molecular weights. Western blot analyses have revealed three distinct anti-TMEM26-reactive protein bands: a ~53 kDa band (p53), a ~44 kDa band (p44), and a ~40 kDa band (p40) . These appear to represent different isoforms of TMEM26, as validated through peptide blocking experiments. Breast cancer cells express both non-glycosylated and N-glycosylated forms of TMEM26 . When selecting antibodies, researchers should consider which epitope is targeted, as some antibodies specifically recognize the C-terminal region of TMEM26 .

How should researchers approach subcellular fractionation to study TMEM26?

Due to TMEM26's membrane localization, subcellular fractionation is recommended when studying this protein. Researchers should prepare at least three separate protein fractions for comprehensive analysis:

• Plasma membrane fraction - crucial for detecting the primary localization of TMEM26

• Cytosolic fraction - to detect any non-membrane-bound forms

• Nuclear fraction - to identify potential nuclear translocation events

This approach has successfully demonstrated TMEM26 presence predominantly in plasma membrane fractions of multiple cancer cell lines . When performing Western blot analysis, researchers should optimize protein loading to detect all potential isoforms, as some may be expressed at lower levels depending on cell type.

What are the optimal techniques for validating TMEM26 antibody specificity?

Antibody validation is critical for TMEM26 research. The following methodological approaches are recommended:

• Peptide competition assay: Pre-incubate the TMEM26 antibody with the same peptide (PrEST antigen TMEM26) used to generate the antibody. This should abolish detection of TMEM26 bands in Western blot, confirming specificity. Include an unrelated antibody (e.g., anti-Elf-1) as a control to demonstrate that the blocking effect is specific to TMEM26 .

• RNAi depletion: Compare antibody signal in TMEM26-expressing cells versus cells where TMEM26 has been depleted via RNAi. This approach verifies that the detected signal is indeed TMEM26 .

• Multiple antibody approach: Use antibodies recognizing different epitopes of TMEM26 (e.g., N-terminal versus C-terminal) to confirm findings.

What applications are suitable for TMEM26 antibodies and what are their specific requirements?

TMEM26 antibodies have been successfully employed in multiple applications with the following methodological considerations:

How should researchers prepare tissue samples for optimal TMEM26 detection in immunohistochemistry?

For immunohistochemical detection of TMEM26, the following protocol has been effectively implemented:

• Fix tissue sections with 3% hydrogen peroxide for 15 minutes

• Wash with Phosphate-Buffered Saline (PBS) three times at room temperature

• Incubate with primary TMEM26 antibodies at 37°C for 30 minutes followed by 4°C overnight

• Apply secondary antibody and incubate for 30 minutes at 37°C

• Incubate with HRP-labeled avidin for 30 minutes at 37°C

• React with domain antibodies for 3–10 minutes before stopping the reaction with ddH₂O

• Counterstain with hematoxylin followed by dehydration

This methodology has successfully detected TMEM26 expression in ESCC tumors versus normal tissues.

How can researchers investigate TMEM26's role in epithelial-mesenchymal transition (EMT)?

To study TMEM26's involvement in EMT, researchers should implement a multi-technique approach:

• Modulate TMEM26 expression: Use RNAi for depletion in TMEM26-high cell lines or overexpression systems in TMEM26-low cell lines

• Assess EMT markers: Examine expression of epithelial markers (E-cadherin, ZO-1) and mesenchymal markers (N-cadherin, vimentin) via Western blot and immunofluorescence

• Functional assays: Perform wound healing and Transwell migration/invasion assays to quantify EMT-related cellular behaviors

• Signaling pathway analysis: Investigate NF-κB pathway activation through nuclear translocation of p65 and phosphorylation of IκBα

Studies in ESCC have demonstrated that TMEM26 depletion suppresses EMT-related alterations while overexpression promotes these changes, without affecting cell growth .

What approaches should be used to examine TMEM26's impact on tight junction formation?

To investigate TMEM26's role in disrupting tight junctions:

• Tight junction protein localization: Use immunofluorescence to visualize plasma membrane localization and assembly of tight junction proteins (claudins, occludin, ZO-1)

• Barrier function assays: Measure transepithelial electrical resistance (TEER) and paracellular permeability using fluorescent tracers

• Protein-protein interaction studies: Perform co-immunoprecipitation to identify TMEM26's interaction partners within tight junction complexes

• Live-cell imaging: Monitor tight junction dynamics in real-time using fluorescently tagged junction proteins

Research has shown that TMEM26 can impair the plasma membrane presentation and assembly of tight junction proteins, providing a mechanism for its role in EMT and metastasis .

How can researchers effectively study TMEM26 in metastatic models?

For investigating TMEM26's role in metastasis:

• In vivo metastasis models: Implement liver metastatic murine models with TMEM26-modulated cells to assess metastatic potential

• Circulating tumor cell analysis: Examine TMEM26 expression in CTCs versus primary tumors

• Metastatic tissue analysis: Compare TMEM26 levels between primary tumors and metastatic lesions using immunohistochemistry

• Multiple cell line comparison: Analyze TMEM26 expression across cell lines with different metastatic potentials

Animal studies have confirmed TMEM26's contributive role in metastatic ESCC, providing evidence for its potential as a therapeutic target .

What glycosylation considerations are important when studying TMEM26?

TMEM26 undergoes N-glycosylation, which can impact antibody detection and protein function:

• Deglycosylation experiments: Treat samples with glycosidases (PNGase F) before Western blotting to distinguish between glycosylated and non-glycosylated forms

• Glycosylation site prediction: Use bioinformatic tools to identify potential N-glycosylation sites

• Glycosylation site mutants: Generate mutants at predicted glycosylation sites to study functional impact

• Cell-type specific glycosylation: Compare glycosylation patterns across different tissues/cell types

Breast cancer cells have been shown to express both non-glycosylated and N-glycosylated forms of TMEM26, highlighting the importance of this post-translational modification .

What are common challenges in TMEM26 antibody applications and how can they be addressed?

How can researchers distinguish between TMEM26 isoforms in their experiments?

To effectively differentiate between TMEM26 isoforms:

• Isoform-specific antibodies: Use antibodies that target unique regions of specific isoforms

• High-resolution gel systems: Employ gradient gels for better separation of closely sized isoforms

• Mass spectrometry: Confirm isoform identity through peptide sequencing

• RT-PCR with isoform-specific primers: Correlate protein expression with transcript variants

• Recombinant isoform expression: Use recombinant TMEM26 isoforms as positive controls

Research has identified multiple TMEM26 reactive bands (p40, p44, p53) that can be confirmed as TMEM26 isoforms through peptide blocking experiments .

How should researchers approach TMEM26 studies in retinal tissue?

For investigating TMEM26 in retinal contexts:

• Layer-specific analysis: Focus on Inner Plexiform Layer (IPL) and Outer Plexiform Layer (OPL) where TMEM26 is predominantly expressed

• Co-localization studies: Combine TMEM26 antibodies with markers for specific retinal cell types

• Comparative expression analysis: Compare TMEM26 levels between normal and glaucomatous retinas

• Functional studies: Investigate TMEM26 knockdown/overexpression effects on retinal cell function

TMEM26 shows specific expression in the IPL and OPL of the retina, with potential weak association with Primary Open-Angle Glaucoma (POAG) .

What controls are essential when studying TMEM26 in disease contexts?

When investigating TMEM26 in disease models, include these critical controls:

• Tissue-matched controls: Compare diseased tissue with appropriate normal tissue from the same anatomical location

• Isotype controls: Use matched isotype antibodies to assess non-specific binding

• Peptide competition controls: Confirm signal specificity through peptide blocking

• Positive expression controls: Include tissues/cells known to express TMEM26

• Multiple antibody validation: Confirm findings with antibodies targeting different TMEM26 epitopes

Studies in ESCC have effectively employed these controls to demonstrate elevated TMEM26 expression in tumors compared to adjacent non-cancerous tissues .

How can researchers quantitatively assess TMEM26 expression differences between normal and disease states?

For rigorous quantitative analysis of TMEM26 expression:

• Digital image analysis: Use software to quantify immunohistochemistry staining intensity

• Western blot densitometry: Perform densitometric analysis normalized to appropriate loading controls

• Flow cytometry: Quantify per-cell expression levels across populations

• qRT-PCR correlation: Correlate protein levels with transcript abundance

• Large cohort analysis: Include sufficient sample numbers for statistical power

Research has successfully used these approaches to demonstrate higher TMEM26 expression in ESCC samples compared to non-cancerous tissues, correlating with invasive and metastatic properties .