Recombinant Human Transmembrane protein 61 (TMEM61)

What cellular membranes contain TMEM61?

Based on current research, TMEM61's specific membrane localization has not been definitively characterized. Unlike some other TMEM family members that have well-defined localizations (such as TMEM45B in the endoplasmic reticulum, trans-Golgi, endosomes, and lysosomes), TMEM61's precise subcellular distribution requires further investigation . Researchers studying TMEM61 localization should consider employing immunofluorescence microscopy with co-localization markers for various cellular compartments (plasma membrane, ER, Golgi, nuclear membrane, etc.) using validated TMEM61 antibodies .

What are the recommended methods for detecting TMEM61 in biological samples?

The primary validated method for detecting TMEM61 in biological samples is immunohistochemistry (IHC) . When performing IHC for TMEM61 detection:

• Use a validated antibody such as 20997-1-AP at a dilution of 1:50-1:500

• For antigen retrieval, use TE buffer at pH 9.0 (alternatively, citrate buffer at pH 6.0 may be used)

• Positive control: Human kidney tissue has shown detectable TMEM61 expression

Other potential detection methods that could be optimized include:

• Western blotting (using the same antibody)

• Real-time PCR for mRNA expression analysis

• RNA-seq for transcriptomic profiling

What is the expression pattern of TMEM61 in normal human tissues?

TMEM61 has been detected in human kidney tissue through immunohistochemistry . For comprehensive expression profiling, researchers should consider:

• Analyzing public transcriptomic databases (GTEx, Human Protein Atlas)

• Performing tissue microarray analysis using validated TMEM61 antibodies

• Conducting qRT-PCR across a panel of normal human tissues

No comprehensive expression atlas specific to TMEM61 was provided in the search results, indicating a potential knowledge gap that researchers might address.

How should researchers validate TMEM61 antibody specificity?

For robust TMEM61 detection, antibody validation is crucial. Recommended validation approaches include:

• Positive and negative control tissues (kidney tissue as positive control)

• Peptide competition assays to confirm binding specificity

• TMEM61 knockdown or knockout controls to confirm signal specificity

• Western blot analysis to confirm detection of a single band at the expected molecular weight (22 kDa)

• Testing multiple antibodies targeting different epitopes of TMEM61

What is known about TMEM61's role in cancer biology?

While specific functions of TMEM61 in cancer have not been extensively characterized, it has been studied in the context of head and neck squamous cell carcinoma (HNSCC) . In HNSCC, TMEM61 showed negative correlations with several other TMEM family members, including TMEM97, ANO1, TMEM45B, TMEM140, and TMEM173 .

To investigate TMEM61's role in cancer:

• Analyze TMEM61 expression in tumor vs. normal tissues using TCGA and GEO datasets

• Perform knockdown/overexpression studies in relevant cancer cell lines

• Assess effects on hallmark cancer phenotypes (proliferation, migration, invasion, apoptosis)

• Investigate correlations with patient survival and clinical parameters

• Employ pathway analysis to identify associated signaling networks

Unlike some other TMEM family members that have established roles as oncogenes or tumor suppressors (e.g., TMEM45B, TMEM119, TMEM48), TMEM61's specific function in cancer progression requires further investigation .

How does TMEM61 expression correlate with other TMEM family members?

TMEM61 shows interesting correlation patterns with other TMEM family members in HNSCC. Specifically, TMEM61 exhibits negative correlations with TMEM97, ANO1, TMEM45B, TMEM140, and TMEM173 . This suggests potential regulatory relationships or participation in opposing biological processes.

For researchers investigating these correlations:

• Analyze co-expression patterns across multiple cancer types and normal tissues

• Perform perturbation experiments (knockdown/overexpression of one TMEM to assess effects on others)

• Investigate shared transcriptional regulators or miRNAs targeting multiple TMEM genes

• Conduct protein-protein interaction studies to identify possible physical associations

• Perform pathway analysis to determine if negatively correlated TMEMs participate in opposing cellular processes

What experimental approaches are recommended for studying TMEM61 function?

To elucidate TMEM61 function, consider these methodological approaches:

• Gene Modulation Studies:

  • siRNA or shRNA knockdown

  • CRISPR/Cas9 knockout

  • Overexpression of tagged TMEM61 (consider C- or N-terminal tags based on predicted topology)

• Interaction Studies:

  • Co-immunoprecipitation to identify binding partners

  • Proximity labeling (BioID or APEX2) to identify neighboring proteins

  • Yeast two-hybrid screening

• Functional Assays:

  • For cancer research: proliferation, migration, invasion, and apoptosis assays

  • For immunological research: immune cell co-culture systems (given correlations with immune-related TMEMs)

• Bioinformatic Analyses:

  • Correlation with hallmark gene sets

  • Protein domain analysis and evolutionary conservation

  • Single-cell RNA-seq to identify cell populations expressing TMEM61

How might post-translational modifications affect TMEM61 function?

While specific post-translational modifications (PTMs) of TMEM61 have not been extensively characterized in the provided search results, investigating potential PTMs is valuable for understanding protein regulation. Researchers should consider:

• Prediction tools for potential modification sites:

  • Phosphorylation: NetPhos, GPS

  • Glycosylation: NetNGlyc, NetOGlyc

  • Ubiquitination: UbPred

  • SUMOylation: GPS-SUMO

• Experimental approaches:

  • Mass spectrometry to identify modifications

  • Mutation of predicted modification sites

  • Western blotting with phospho-specific antibodies

  • Treatment with deglycosylation enzymes

• Functional impact assessment:

  • Comparing wild-type vs. modification-site mutants in localization assays

  • Evaluating effects on protein stability and turnover

  • Assessing impact on protein-protein interactions

What is the potential relationship between TMEM61 and immune responses?

While the direct relationship between TMEM61 and immune responses has not been fully characterized, several other TMEM family members show immune-related functions. For example, TMEM173 (also known as STING) plays a role in innate immunity, and TMEM61 shows a negative correlation with TMEM173 in HNSCC .

To investigate potential immune-related functions:

• Analyze TMEM61 expression in immune cell subtypes using single-cell RNA-seq datasets

• Correlate TMEM61 expression with immune infiltration scores in tumors

• Assess effects of immune stimulants (IFNs, TLR ligands) on TMEM61 expression

• Investigate consequences of TMEM61 modulation on cytokine production

• Perform co-culture experiments with immune and cancer cells with TMEM61 knockdown/overexpression

What considerations are important when producing recombinant TMEM61 for functional studies?

Production of properly folded recombinant transmembrane proteins presents significant challenges. For TMEM61:

• Expression System Selection:

  • Mammalian cells for proper folding and PTMs

  • Insect cells as an alternative system

  • Bacterial systems with specialized tags (MBP, SUMO) for solubility

• Purification Strategy:

  • Detergent selection critical for maintaining native structure

  • Consider nanodisc or liposome reconstitution

  • Affinity tags placement (avoid disrupting transmembrane domains)

• Quality Control:

  • Circular dichroism to assess secondary structure

  • Size exclusion chromatography to evaluate oligomeric state

  • Functional binding assays if ligands are known

• Alternative Approaches:

  • Cell-free expression systems

  • Truncated constructs excluding transmembrane domains for soluble fragment studies

  • Synthetic peptides for specific domain investigations

How can researchers investigate the role of TMEM61 in disease progression using patient-derived samples?

For translational research on TMEM61 in disease:

• Tissue Microarray Analysis:

  Sample Type	Antibody Dilution	Antigen Retrieval	Scoring Method
  FFPE tissues	1:50-1:500	TE buffer pH 9.0	H-score (intensity × percentage)
  Frozen tissues	1:100-1:200	Not required	Quantitative image analysis

• Correlation with Clinical Parameters:

  • Disease stage

  • Treatment response

  • Survival outcomes

  • Molecular subtypes

• Multi-omics Integration:

  • Correlate protein expression with genomic alterations

  • Assess mRNA-protein correlation

  • Identify pathways co-regulated with TMEM61

• Ex vivo Functional Studies:

  • Patient-derived organoids with TMEM61 modulation

  • Ex vivo drug sensitivity testing based on TMEM61 status

What computational tools are most useful for predicting TMEM61 interactions and functions?

Given limited experimental characterization, computational approaches can guide TMEM61 research:

• Protein Structure Prediction:

  • AlphaFold2 or RoseTTAFold for 3D structure prediction

  • TMHMM or TOPCONS for transmembrane topology

• Interaction Prediction:

  • STRING database for potential protein-protein interactions

  • STITCH for protein-chemical interactions

  • PrePPI for structure-based interaction prediction

• Functional Annotation:

  • Gene Ontology enrichment of co-expressed genes

  • Domain-based function prediction

  • Evolutionary analysis for conserved functional regions

• Expression Correlation Tools:

  • GEPIA2 for correlation analysis across TCGA tumors

  • Co-expression networks (WGCNA) to identify TMEM61 modules

  • GSEA for pathway enrichment based on correlations

What controls should be included in TMEM61 expression studies?

For robust TMEM61 research, these controls are essential:

• Antibody Validation Controls:

  • Positive tissue control (kidney)

  • Blocking peptide control

  • Secondary antibody-only control

• Expression Analysis Controls:

  • Multiple reference genes for qRT-PCR (GAPDH, ACTB, 18S rRNA)

  • Tissue-matched normal samples

  • Cell lines with known TMEM61 expression levels

• Functional Study Controls:

  • Multiple siRNA/shRNA sequences targeting different regions

  • Non-targeting control siRNA/shRNA

  • Empty vector controls for overexpression

  • Rescue experiments with siRNA-resistant constructs

What are the recommended protocols for TMEM61 immunohistochemistry?

Based on the antibody information provided :

• Sample Preparation:

  • Formalin-fixed paraffin-embedded (FFPE) tissue sections (4-6 μm)

  • Deparaffinization and rehydration through graded alcohols

• Antigen Retrieval:

  • Primary method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval (pressure cooker or microwave)

• Staining Protocol:

  • Endogenous peroxidase blocking: 3% H₂O₂

  • Protein blocking: 5% normal goat serum

  • Primary antibody: Anti-TMEM61 (20997-1-AP) at 1:50-1:500 dilution

  • Incubation: Overnight at 4°C

  • Detection system: HRP-polymer and DAB substrate

  • Counterstain: Hematoxylin

• Evaluation Methods:

  • H-score (intensity × percentage)

  • Digital image analysis for quantification

  • Expert pathologist assessment

How can researchers address the challenge of studying low-abundance transmembrane proteins like TMEM61?

Transmembrane proteins often present detection challenges due to low abundance and hydrophobicity:

• Enrichment Strategies:

  • Membrane fraction isolation before Western blotting

  • Immunoprecipitation for concentration

  • Proximity labeling to amplify signal

• Detection Enhancement:

  • Signal amplification systems (TSA, polymer-based detection)

  • Super-resolution microscopy for localization studies

  • Targeted mass spectrometry (PRM/MRM)

• Expression Systems:

  • Inducible expression systems for controlled overexpression

  • Viral transduction for difficult-to-transfect cells

  • Fusion with fluorescent proteins for live imaging

• Transcriptomic Approaches:

  • Digital droplet PCR for low-abundance transcripts

  • RNA-FISH for single-cell detection

  • Bulk RNA-seq with deep sequencing

What are common challenges in TMEM61 antibody-based detection and how can they be addressed?

Researchers may encounter several issues when using antibodies to detect TMEM61:

• High Background:

  • Increase blocking time/concentration

  • Optimize antibody dilution (try 1:100-1:500 range)

  • Increase washing steps duration/frequency

  • Use alternative blocking agents (BSA, casein, commercial blockers)

• Weak or No Signal:

  • Try alternative antigen retrieval methods (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Increase antibody concentration carefully

  • Extend primary antibody incubation time

  • Use signal amplification systems

• Non-specific Binding:

  • Pre-adsorb antibody with tissue powder

  • Validate with TMEM61 knockdown controls

  • Test multiple antibodies targeting different epitopes

  • Include peptide competition controls

• Inconsistent Results:

  • Standardize tissue processing protocols

  • Control fixation time and conditions

  • Use automated staining platforms

  • Incorporate internal control samples in each run

How should researchers interpret contradictory findings about TMEM61 function?

When faced with conflicting results regarding TMEM61:

• Context-Dependent Effects:

  • Consider cell/tissue type differences

  • Evaluate impact of microenvironment

  • Assess experimental conditions (2D vs. 3D, in vitro vs. in vivo)

• Technical Considerations:

  • Compare antibody specificities and epitopes

  • Evaluate knockdown efficiency and specificity

  • Assess overexpression levels (physiological vs. non-physiological)

• Analytical Approaches:

  • Meta-analysis of multiple studies

  • Stratification by molecular subtypes

  • Single-cell analysis to identify cell-specific effects

• Reconciliation Strategies:

  • Design experiments to directly test contradictions

  • Investigate potential dual functions in different contexts

  • Consider protein isoforms or post-translational modifications

What statistical approaches are recommended for analyzing TMEM61 expression data in clinical samples?

For robust statistical analysis:

What are promising research areas for understanding TMEM61 function?

Based on current knowledge gaps, these research directions could advance TMEM61 understanding:

• Structural Biology:

  • Cryo-EM structure determination

  • Molecular dynamics simulations

  • Structure-function relationship studies

• Systems Biology:

  • Multi-omics integration

  • Network analysis of TMEM protein interactions

  • Comparative analysis across TMEM family members

• Disease Associations:

  • Cancer progression and metastasis

  • Potential roles in immune pathologies

  • Correlation with treatment response

• Evolutionary Perspective:

  • Cross-species conservation analysis

  • Paralog functional divergence

  • Selective pressure analysis

How might TMEM61 interact with other transmembrane proteins in functional complexes?

Investigating TMEM61's role in protein complexes:

• Protein Complex Identification:

  • Blue native PAGE for membrane complexes

  • Cross-linking mass spectrometry

  • Co-immunoprecipitation with mild detergents

  • FRET/BRET for proximity detection

• Complex Formation Dynamics:

  • Live-cell imaging with fluorescently tagged proteins

  • Inducible dimerization systems

  • Hydrogen-deuterium exchange mass spectrometry

• Functional Impact:

  • Mutagenesis of interaction interfaces

  • Dominant-negative approaches

  • Competitive inhibition with peptides

• Bioinformatic Prediction:

  • Coevolution analysis

  • Interface prediction algorithms

  • Assembly prediction from AlphaFold multimers