TMEM56 Antibody

What is TMEM56 and why is it studied?

TMEM56 (Transmembrane protein 56) is a cellular membrane protein with approximately 43 kDa molecular weight that spans the cell membrane. It plays significant roles in cellular signaling pathways, membrane trafficking, and protein transport mechanisms. The protein's versatile functions make it a target for investigations into biological processes including cell growth, differentiation, and apoptosis. Research involving TMEM56 is essential for understanding its impact on cellular physiology and identifying potential therapeutic strategies for disorders associated with dysregulated protein transport and membrane dynamics .

What types of TMEM56 antibodies are available for research?

TMEM56 antibodies are predominantly available as rabbit polyclonal antibodies that target human TMEM56. These antibodies are typically generated against recombinant human TMEM56 protein fragments, such as the C-terminal region or specific amino acid sequences (e.g., 232-263AA). Most commercial antibodies are unconjugated, though some vendors offer conjugated versions with HRP, FITC, or biotin for specialized applications. The antibodies undergo protein G purification to ensure high specificity and are typically supplied in liquid form with glycerol-based storage buffers .

What are the validated applications for TMEM56 antibodies?

TMEM56 antibodies have been validated for multiple research applications, primarily:

• Immunohistochemistry of paraffin-embedded tissues (IHC-P): Typically used at dilutions of 1:20-1:500

• Immunofluorescence (IF): Generally effective at dilutions of 1:50-1:200

• Western blotting (WB): For protein detection in cell and tissue lysates

• ELISA: Used at dilutions of 1:2000-1:10000

These antibodies have specifically demonstrated reactivity with human samples, including normal tissues and disease models such as glioma, endometrial cancer, and other tumors .

What is the recommended storage protocol for TMEM56 antibodies?

To maintain antibody integrity and performance, TMEM56 antibodies should be stored according to a two-tier approach. For short-term use (up to 2 weeks), refrigeration at 2-8°C is sufficient. For long-term storage, antibodies should be kept at -20°C in small aliquots to prevent freeze-thaw cycles that can degrade antibody quality. Most commercial preparations contain preservatives (such as 0.03% Proclin 300) and stabilizers (typically 50% glycerol in PBS, pH 7.4) to maintain antibody function during storage. When handling, it's advisable to avoid repeated freeze-thaw cycles by creating single-use aliquots before freezing .

How should I optimize TMEM56 antibody dilutions for different applications?

Optimizing antibody dilutions requires systematic titration based on the specific application:

For each new tissue type or cell line, perform a preliminary experiment using a dilution series within the recommended range. Evaluate results based on signal specificity, background levels, and signal-to-noise ratio. Include appropriate positive and negative controls to validate specificity. When working with new sample types, broader titration ranges may be necessary to determine optimal conditions .

What controls should I include when using TMEM56 antibodies?

A robust experimental design with TMEM56 antibodies requires multiple control types:

• Positive tissue controls: Human tissues known to express TMEM56, such as glioma, endometrial tissue, or HeLa cells that have been validated in previous studies

• Negative controls:

  • Primary antibody omission: Replace primary antibody with antibody diluent

  • Isotype control: Use non-specific rabbit IgG at the same concentration

  • Blocking peptide: Pre-incubate antibody with immunizing peptide to demonstrate binding specificity

• Technical controls:

  • Loading control for Western blots (e.g., β-actin, GAPDH)

  • Nuclear counterstain (e.g., DAPI) for immunofluorescence to help localize cellular structures

These controls help distinguish between specific TMEM56 signals and non-specific background, particularly important when analyzing new tissue types or when troubleshooting unexpected results .

How do I prepare samples for optimal TMEM56 detection in IHC?

For optimal TMEM56 detection in immunohistochemistry, follow this validated protocol:

• Fixation: Fix tissues in 10% neutral-buffered formalin for 24-48 hours, depending on tissue size

• Embedding and sectioning: Process tissues through graded alcohols and xylene, embed in paraffin, and section at 4-5μm thickness

• Antigen retrieval: Critical step for transmembrane proteins like TMEM56

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes is most commonly effective

  • Alternative: Tris-EDTA buffer (pH 9.0) if citrate buffer yields weak signals

• Blocking: Block with 3-5% normal serum (from the same species as the secondary antibody) for 30-60 minutes

• Primary antibody incubation: Apply TMEM56 antibody at optimal dilution (typically 1:100) and incubate overnight at 4°C

• Detection system: Use appropriate detection system (HRP/DAB or fluorescence-based) following manufacturer's protocols

• Counterstaining: Hematoxylin for brightfield or DAPI for fluorescence

The protocol has been validated in human tissues including glioma, uterine tissue, and endometrial cancer samples, demonstrating specific membrane and cytoplasmic staining patterns .

What are common troubleshooting strategies for weak or absent TMEM56 staining?

When encountering weak or absent TMEM56 staining, systematically address these potential issues:

• Antibody factors:

  • Verify antibody integrity and storage conditions

  • Try increasing antibody concentration (use a titration series)

  • Consider longer incubation times (overnight at 4°C instead of 1-2 hours at room temperature)

  • Test alternative TMEM56 antibody clones targeting different epitopes

• Sample factors:

  • Optimize fixation duration (overfixation can mask epitopes)

  • Test alternative antigen retrieval methods (pH 6.0 vs. pH 9.0 buffers, or enzymatic retrieval)

  • Verify sample handling and storage conditions

  • Ensure target protein isn't degraded in your samples

• Detection system:

  • Use signal amplification methods (e.g., avidin-biotin complex, tyramide signal amplification)

  • Ensure secondary antibody compatibility with primary antibody species

  • Check detection substrate freshness and activity

• Expression levels:

  • Verify TMEM56 expression in your sample type through alternative methods (qPCR, RNA-seq)

  • Include positive control samples with known TMEM56 expression

These approaches have successfully resolved detection issues in various tissue types including glioma and endometrial tissues where TMEM56 staining can be particularly challenging .

How can I minimize background when using TMEM56 antibodies?

High background signal can compromise TMEM56 detection specificity. Implement these validated strategies to improve signal-to-noise ratio:

• Blocking optimization:

  • Increase blocking time (from 30 to 60 minutes)

  • Test different blocking reagents (BSA, normal serum, commercial blocking solutions)

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization

• Antibody considerations:

  • Further dilute primary and secondary antibodies

  • Reduce antibody incubation times

  • Use affinity-purified antibodies (all commercial TMEM56 antibodies are protein G purified)

  • Pre-absorb antibodies with tissue powder or non-specific proteins

• Washing steps:

  • Increase wash duration and number of washes

  • Include detergents (0.05-0.1% Tween-20) in wash buffers

  • Use PBS-T (PBS with 0.05% Tween-20) for more stringent washing

• Tissue-specific treatments:

  • Block endogenous peroxidase (3% H₂O₂, 10-15 minutes) before antibody application

  • For tissues with high biotin content, use avidin-biotin blocking kit

  • Consider autofluorescence quenching reagents for fluorescence applications

These optimization strategies have proven effective across multiple tissue types, particularly in reducing non-specific binding in human glioma samples where background can be problematic .

What specific considerations apply to TMEM56 detection in immunofluorescence?

Successful TMEM56 detection in immunofluorescence requires specific technical considerations:

• Cell preparation:

  • For adherent cells (e.g., HeLa), culture on glass coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 for 10 minutes to access transmembrane epitopes

• TMEM56 antibody application:

  • Dilute to 1:50-1:200 range (most publications use 1:100)

  • Incubate in humidity chamber overnight at 4°C to maximize sensitivity

  • Include 1% BSA in antibody diluent to reduce background

• Detection and visualization:

  • Use fluorophore-conjugated secondary antibodies (Alexa Fluor 488 is commonly used)

  • Counterstain with DAPI (1μg/ml, 5 minutes) for nuclear visualization

  • Mount with anti-fade mounting medium to prevent photobleaching

• Imaging optimization:

  • Use confocal microscopy for precise subcellular localization

  • Adjust exposure settings to prevent signal saturation

  • Capture Z-stacks for transmembrane proteins to visualize complete membrane distribution

Successful immunofluorescence detection of TMEM56 has been demonstrated in HeLa cells, showing characteristic membrane and cytoplasmic localization patterns when following these guidelines .

How can I utilize TMEM56 antibodies for co-localization studies?

TMEM56 co-localization studies require sophisticated experimental design to reveal functional relationships with other proteins:

• Multi-channel immunofluorescence protocol:

  • Select TMEM56 antibody and antibodies against potential interaction partners

  • Ensure primary antibodies are raised in different host species (e.g., rabbit anti-TMEM56 and mouse anti-partner protein)

  • Use spectrally distinct fluorophores for secondary antibodies (e.g., Alexa 488 for TMEM56, Alexa 594 for partner)

  • Include appropriate controls: single-antibody stains and isotype controls

• Target selection strategy: Based on TMEM56's known functions, consider these co-localization candidates:

  • Membrane trafficking markers: Rab GTPases, clathrin, caveolin

  • Endosomal compartment markers: EEA1 (early endosomes), LAMP1 (lysosomes)

  • Other transmembrane proteins implicated in cellular signaling

• Analysis methodology:

  • Collect images using confocal microscopy with sequential scanning to prevent bleed-through

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Perform pixel intensity correlation analysis across multiple cells (n>30)

  • Consider super-resolution microscopy for refined spatial analysis of membrane proteins

This approach has proven effective in elucidating the relationships between transmembrane proteins and their functional partners in cellular signaling and trafficking pathways, though specific TMEM56 co-localization data is still emerging in the literature .

What is the optimal approach for using TMEM56 antibodies in flow cytometry?

While not explicitly validated in the provided search results, flow cytometry analysis of TMEM56 can be implemented following these research-based guidelines:

• Cell preparation optimization:

  • Use gentle cell dissociation methods (e.g., enzyme-free dissociation buffer) to preserve membrane proteins

  • For intracellular epitopes, permeabilize with 0.1% saponin rather than harsher detergents

  • Maintain cells at 4°C throughout processing to prevent internalization of surface proteins

• Antibody application protocol:

  • Test both direct and indirect labeling approaches

  • For indirect labeling: Use primary TMEM56 antibody at 1:50-1:100 dilution, followed by fluorophore-conjugated secondary antibody

  • For direct approach: Consider conjugated TMEM56 antibodies (FITC, PE) if available

  • Include FcR blocking reagent to reduce non-specific binding

• Controls and validation:

  • Use isotype control matched to TMEM56 antibody subclass

  • Include fluorescence-minus-one (FMO) controls

  • Validate specificity through TMEM56 knockdown/knockout cells

  • Compare expression patterns with immunofluorescence data on the same cell types

• Data analysis considerations:

  • Gate on viable single cells

  • Compare median fluorescence intensity (MFI) rather than percent positive

  • Normalize to isotype control

  • Consider comparing expression across different cell states (e.g., differentiation, activation)

This methodological approach integrates best practices for membrane protein detection in flow cytometry and can be adapted specifically for TMEM56 analysis .

How can I design experiments to investigate TMEM56 function using antibody-based approaches?

Functional investigation of TMEM56 using antibody-based approaches requires sophisticated experimental design:

• Protein interaction studies:

  • Co-immunoprecipitation: Use TMEM56 antibody to pull down protein complexes, then identify binding partners through mass spectrometry

  • Proximity ligation assay (PLA): Combine TMEM56 antibody with antibodies against suspected interaction partners to visualize protein-protein interactions in situ

  • FRET/FLIM analysis: Use fluorophore-conjugated antibodies to measure energy transfer between TMEM56 and potential partners

• Functional blocking experiments:

  • Apply TMEM56 antibodies to live cells to potentially block protein function

  • Assess phenotypic changes in:

    • Membrane trafficking (using fluorescent cargo tracking)

    • Cell migration (wound healing assays)

    • Signal transduction (phosphorylation of downstream targets)

  • Compare with siRNA knockdown effects to validate findings

• Correlation with disease states:

  • Tissue microarray analysis of TMEM56 expression across multiple disease tissues

  • Quantify expression level changes using digital pathology approaches

  • Correlate with clinical outcomes and other molecular markers

  • Compare with gene expression data for integrated analysis

These experimental approaches leverage the specificity of TMEM56 antibodies to investigate functional roles beyond simple protein detection, providing mechanistic insights into TMEM56's cellular functions .

How should I quantify and interpret TMEM56 immunostaining in tissue samples?

Robust quantification of TMEM56 immunostaining requires standardized methods:

• Immunohistochemistry quantification approaches:

  • Semi-quantitative scoring: Use established H-score method (intensity × percentage positive cells)

  • Digital image analysis: Use software algorithms to quantify DAB staining intensity and distribution

  • Specify subcellular localization (membrane, cytoplasmic, or both)

  • Compare staining patterns across different tissue regions (e.g., tumor center vs. periphery)

• Immunofluorescence quantification methods:

  • Measure mean fluorescence intensity (MFI) in defined cellular compartments

  • Assess co-localization coefficients when performing dual staining

  • Quantify percentage of positive cells in heterogeneous populations

  • Use specialized software (ImageJ, CellProfiler) with consistent thresholding parameters

• Interpretation guidelines:

  • Compare with established markers of cellular compartments

  • Consider physiological context and tissue-specific expression patterns

  • Evaluate whether staining pattern is consistent with transmembrane protein localization

  • Correlate with other experimental data (Western blot, qPCR) to validate observations

• Statistical analysis:

  • Use appropriate statistical tests based on data distribution

  • Include sufficient biological replicates (typically n≥3)

  • Report variability measures (standard deviation, standard error)

  • Consider blinded scoring to eliminate observer bias

These approaches have been applied to TMEM56 detection in human glioma, endometrial cancer, and uterine tissues, allowing meaningful comparison of expression patterns across different samples .

What are the expected staining patterns for TMEM56 in different tissue types?

TMEM56 exhibits tissue-specific distribution patterns that must be considered when interpreting results:

• Normal tissue distribution:

  • TMEM56 shows variable expression across human tissues

  • Expected primarily in membranous and cytoplasmic compartments given its identity as a transmembrane protein

  • Particular attention should be paid to epithelial cells, where many transmembrane proteins show enriched expression

• Documented staining patterns:

  • Human uterus (pre-menopause): Moderate membranous and cytoplasmic staining

  • Human glioma: Variable expression with both membranous and cytoplasmic patterns

  • HeLa cells: Distinct membranous staining with some cytoplasmic distribution

  • Endometrial cancer: Often shows altered expression compared to normal endometrial tissue

• Subcellular localization considerations:

  • Primary membranous localization consistent with transmembrane protein function

  • Cytoplasmic staining may represent protein in biosynthetic or trafficking pathways

  • Nuclear staining would be unexpected and should be validated carefully

  • Punctate vesicular patterns may indicate endosomal localization

• Abnormal patterns requiring validation:

  • Exclusively nuclear staining would be considered atypical

  • Complete absence in tissues known to express TMEM56 mRNA

  • Highly heterogeneous expression within a single tissue type

These patterns have been documented through experimental validation using the antibodies described in the search results, particularly in immunohistochemistry and immunofluorescence studies of human tissues .

How do I address contradictory results when using different TMEM56 antibodies?

Contradictory results from different TMEM56 antibodies require systematic investigation:

• Antibody characteristic analysis:

  • Compare epitope regions (N-terminal, C-terminal, internal domains)

  • Assess antibody format (polyclonal vs. monoclonal)

  • Review validation data for each antibody

  • Consider host species and immunization protocols

• Validation experiments:

  • Western blot analysis to confirm specificity and molecular weight

  • Peptide competition assays to verify epitope specificity

  • siRNA knockdown or CRISPR knockout validation

  • Correlation with mRNA expression data (qPCR, RNA-seq)

• Technical variables assessment:

  • Evaluate fixation and preparation protocols

  • Compare antigen retrieval methods

  • Standardize antibody concentrations (use molar equivalents)

  • Test multiple detection systems

• Interpretation framework:

  • Different antibodies may recognize distinct protein isoforms

  • Post-translational modifications might affect epitope accessibility

  • Protein conformation in different cellular compartments could alter antibody binding

  • Tissue-specific factors might influence antibody performance

This systematic approach helps determine whether discrepancies reflect technical issues or biologically meaningful differences in protein expression or modification, ensuring accurate interpretation of TMEM56 distribution and function .

What are emerging applications for TMEM56 antibodies in disease research?

TMEM56 antibodies are finding expanding applications in disease-focused research:

• Cancer research applications:

  • Expression profiling across tumor types (glioma and endometrial cancer already documented)

  • Correlation with tumor grade, stage, and patient outcomes

  • Investigation of TMEM56's potential role in cancer cell signaling and membrane dynamics

  • Biomarker development for diagnostic or prognostic applications

• Neurological disorder investigations:

  • Analysis of TMEM56 distribution in neurological tissue samples

  • Investigation of potential roles in neuronal membrane trafficking

  • Correlation with other transmembrane proteins implicated in neurological disorders

• Cellular physiology studies:

  • Protein transport and membrane organization research

  • Cell differentiation and development investigations

  • Stress response and cellular adaptation mechanisms

• Methodological advancements:

  • Integration with emerging spatial transcriptomics approaches

  • Combination with super-resolution microscopy techniques

  • Use in high-content screening applications

These emerging applications build upon the established roles of TMEM56 in cellular signaling pathways, membrane trafficking, and protein transport mechanisms, expanding our understanding of its functions in normal physiology and disease states .

What quality control measures should be implemented for long-term TMEM56 antibody studies?

For reliable results in longitudinal TMEM56 research, implement these quality control measures:

• Antibody validation program:

  • Regular specificity testing through Western blot analysis

  • Batch-to-batch comparison when reordering antibodies

  • Validation across multiple applications (IHC, IF, WB) to ensure consistent performance

  • Documentation of positive control tissues or cell lines for each application

• Storage and handling protocol:

  • Maintain detailed antibody inventory with dates, lot numbers, and aliquot tracking

  • Store according to manufacturer recommendations (typically -20°C in small aliquots)

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Monitor preservative and storage buffer composition

• Experimental standardization:

  • Maintain consistent fixation and processing protocols

  • Standardize antibody dilutions and incubation conditions

  • Include technical and biological replicates in all experiments

  • Incorporate positive and negative controls in each experimental run

• Data management system:

  • Document all experimental parameters in laboratory information systems

  • Create a central repository of validated protocols and results

  • Implement statistical quality control measures to track antibody performance over time

  • Establish acceptance criteria for control samples

How might TMEM56 antibody research evolve with advancing technologies?

The future landscape of TMEM56 antibody applications will likely evolve with several technological advancements:

• Integration with emerging imaging technologies:

  • Super-resolution microscopy (STORM, PALM, STED) for nanoscale localization of TMEM56

  • Expansion microscopy for enhanced visualization of membrane protein organization

  • Light sheet microscopy for 3D tissue analysis with minimal photobleaching

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

• Single-cell analysis applications:

  • Coupling with single-cell transcriptomics for multi-omic profiling

  • Mass cytometry (CyTOF) incorporation for high-dimensional protein analysis

  • Spatial transcriptomics integration to correlate protein localization with gene expression

  • Microfluidic approaches for dynamic analysis of membrane protein trafficking

• Functional probing methodologies:

  • Optogenetic integration for real-time manipulation of TMEM56 function

  • CRISPR-based screening combined with antibody detection

  • Live-cell antibody-based biosensors for dynamic functional studies

  • Antibody-drug conjugates for targeted functional manipulation

• Artificial intelligence applications:

  • Machine learning algorithms for automated quantification of staining patterns

  • Deep learning approaches for predictive modeling of protein interactions

  • Computer vision techniques for high-throughput image analysis

  • Integrative data analysis frameworks combining antibody-based data with other omics