TMEM256 Antibody

What is TMEM256 protein and what are its key features?

TMEM256 (Transmembrane protein 256) is a human membrane protein also known as C17orf61 or UPF0451 protein C17orf61. The canonical human TMEM256 protein has 113 amino acid residues with a molecular mass of approximately 11.7 kDa . Its primary subcellular localization is in the membrane, consistent with its designation as a transmembrane protein . TMEM256 belongs to the TMEM256 protein family, with gene orthologs reported in multiple species including mouse, rat, bovine, zebrafish, and chimpanzee .

The protein's function hasn't been extensively characterized in the literature provided, but it appears to be associated with extracellular exosomes based on background information in antibody documentation . This suggests potential roles in intercellular communication or molecular transport, though further research is needed to fully elucidate its biological functions.

What types of TMEM256 antibodies are currently available for research?

The predominant type of TMEM256 antibody available for research is rabbit polyclonal antibody, as evidenced across multiple suppliers . These antibodies are primarily unconjugated, though some fluorescent-conjugated versions (e.g., FITC) are available for specialized applications . The current antibody landscape includes:

While most commercially available TMEM256 antibodies target human proteins, some cross-reactivity with mouse or zebrafish proteins has been reported . These antibodies are generated using various immunogens, including:

• Recombinant human TMEM256 protein fragments (amino acids 30-63)

• Synthetic peptides corresponding to residues near the C-terminal of human TMEM256

How is antibody specificity for TMEM256 determined and validated?

Antibody specificity for TMEM256 is determined through multiple validation approaches. For commercial antibodies, manufacturers typically report that "the antibody detects endogenous levels of total TMEM256 protein" , indicating validation against naturally expressed protein targets rather than just recombinant constructs.

Advanced methods for determining antibody specificity involve computational models that identify different binding modes associated with particular ligands. As discussed in phage display research, high-throughput sequencing combined with computational analysis can disentangle binding modes even when they involve chemically similar ligands . Though not specific to TMEM256, these advanced techniques represent the cutting edge of antibody validation.

For researchers requiring rigorous validation, recommended approaches include:

• Western blot analysis showing bands of appropriate molecular weight

• Positive and negative tissue controls in immunohistochemistry

• Knockdown or knockout verification, where antibody signal disappears when target protein expression is eliminated

• Comparison of staining patterns across multiple antibodies targeting different epitopes of the same protein

What are the optimal applications for TMEM256 antibodies?

TMEM256 antibodies have been validated for several experimental applications with varying degrees of optimization. The most commonly supported applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Most commercial TMEM256 antibodies are validated for ELISA applications , making this a primary choice for quantitative detection of the protein.

• Immunofluorescence (IF): Multiple antibodies support IF applications , allowing for subcellular localization studies of TMEM256.

• Immunohistochemistry (IHC): Several antibodies are specifically optimized for IHC applications , enabling tissue-level detection of TMEM256 expression.

• Western Blotting (WB): Some antibodies support WB applications , though this appears less universal than ELISA or IF applications.

• Immunocytochemistry (ICC): A subset of antibodies supports ICC applications , allowing for cellular-level detection in cultured cells.

When selecting an application, researchers should consider that antibody performance can vary significantly between applications even when technical specifications appear similar. The data suggests that ELISA and IF applications have the most consistent validation across multiple antibody products.

How can TMEM256 antibodies be effectively used in protein microarray experiments?

While not specific to TMEM256 antibodies, protein microarray experiments with antibodies follow methodologies that can be applied to TMEM256 research. For comparative fluorescence assays using antibody microarrays:

• Array Preparation: Print antibodies (including anti-TMEM256) in replicate spots on suitable microarray slides. Typically, 9-12 replicate spots per antibody provide sufficient statistical power for analysis .

• Sample Labeling: Label protein samples containing TMEM256 with fluorescent dyes (commonly Cy3/Cy5 or equivalent fluorophores) . When comparing two samples, differential labeling allows direct comparison on the same array.

• Detection and Quantification: After incubation and washing, measure fluorescence intensity ratios. For quantitative analysis, examine the log10 of the red-to-green ratio (log10(R/G)) as a function of TMEM256 concentration .

• Data Analysis Considerations: Be aware that labeling efficiency can be a significant source of variation in protein microarray experiments . Including internal control proteins in the labeling reaction can help correct for this variation.

The detection limit in microarray formats typically approaches 5-30 ng/ml for many proteins , though specific limits for TMEM256 would require empirical determination.

What protocols are recommended for immunohistochemical detection of TMEM256?

For immunohistochemical detection of TMEM256 in tissue samples, the following protocol framework is recommended based on antibody specifications:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Heat at 95-100°C for 15-20 minutes, then cool to room temperature

• Blocking and Antibody Incubation:

  • Block with 5-10% normal serum in PBS for 1 hour

  • Incubate with TMEM256 antibody at manufacturer-recommended dilution (typically 1:100-1:500)

  • Incubate overnight at 4°C or for 1-2 hours at room temperature

• Detection System:

  • Use an appropriate secondary antibody system compatible with rabbit primary antibodies

  • Visualize using DAB (3,3'-diaminobenzidine) or other suitable chromogen

  • Counterstain with hematoxylin

• Controls:

  • Always include a negative control (primary antibody omitted)

  • When possible, include tissues known to express or lack TMEM256

The antibody's ability to "detect endogenous levels of total TMEM256 protein" makes it suitable for examining native expression patterns in human tissues.

What are common challenges when working with TMEM256 antibodies and how can they be addressed?

When working with TMEM256 antibodies, researchers may encounter several technical challenges that can affect experimental outcomes:

• Signal Specificity Issues:

  • Challenge: Nonspecific binding leading to background signal

  • Solution: Optimize blocking conditions using 3-5% BSA or normal serum matching the species of the secondary antibody. Increase washing duration and frequency with detergent-containing buffer.

• Detection Sensitivity:

  • Challenge: Low signal when detecting endogenous TMEM256

  • Solution: As TMEM256 is a membrane protein (11.7 kDa) , optimize extraction methods using appropriate detergents for membrane proteins. Consider signal amplification systems or longer primary antibody incubation times.

• Epitope Accessibility:

  • Challenge: Limited antibody binding due to protein conformation

  • Solution: For TMEM256 detection in fixed tissues or cells, evaluate multiple antigen retrieval methods, as commercially available antibodies target different epitopes including the C-terminus or amino acids 30-63 .

• Variability in Replicate Experiments:

  • Challenge: Inconsistent results between experiments

  • Solution: Standardize protocols, particularly antibody storage conditions (most TMEM256 antibodies should be stored at -20°C) . For quantitative assays, prepare master mixes and process samples in parallel.

When troubleshooting, always refer to the specific product documentation as formulations vary between suppliers, with some antibodies provided in glycerol-containing buffers (e.g., "50% Glycerol, 0.01M PBS, pH 7.4") which affects dilution calculations.

How should TMEM256 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of TMEM256 antibodies is critical for maintaining their activity and specificity. Based on manufacturer recommendations:

• Storage Temperature:

  • Store TMEM256 antibodies at -20°C for long-term storage

  • Some formulations may allow storage at -80°C for extended periods

• Avoid Freeze-Thaw Cycles:

  • Minimize repeated freeze-thaw cycles which can degrade antibody quality

  • Aliquot antibodies upon initial thawing for single-use portions

• Working Solution Preparation:

  • Dilute antibodies in recommended buffers immediately before use

  • For TMEM256 antibodies in glycerol-containing formulations (e.g., "50% Glycerol, 0.01M PBS, pH 7.4") , bring to room temperature before opening to prevent condensation

• Stability Considerations:

  • Most TMEM256 antibodies are formulated with preservatives such as 0.03% Proclin 300 or 0.05% NaN3 to prevent microbial growth

  • Working dilutions without preservatives should be prepared fresh and not stored for extended periods

Following these guidelines will help ensure consistent antibody performance across experiments and maximize the usable lifespan of TMEM256 antibodies.

How can computational models improve TMEM256 antibody specificity and design?

Advanced computational approaches can significantly enhance TMEM256 antibody specificity and design, particularly when discrimination between very similar epitopes is required. Recent methodological advances include:

• Binding Mode Identification:
  Computational models can identify different binding modes associated with particular ligands, even when these epitopes are chemically very similar . For TMEM256 research, this approach could help design antibodies that specifically distinguish between closely related membrane proteins or between different conformational states of TMEM256.

• High-Throughput Sequencing Integration:
  Combining phage display experiments with high-throughput sequencing and computational analysis provides additional control over antibody specificity profiles . This approach allows:

  • Analysis of selection results beyond just the highest-affinity binders

  • Identification of sequence features associated with specific binding properties

  • Design of novel antibodies with customized specificity profiles

• Customized Specificity Design:
  Computational models can be used to design antibodies with:

  • Specific high affinity for particular target epitopes of TMEM256

  • Cross-specificity for multiple target epitopes when desired

  • Reduced binding to unintended targets

These approaches extend beyond traditional antibody generation methods that rely solely on selection, which is limited by library size and provides less control over specificity profiles .

What are the current limitations in TMEM256 antibody research and future directions?

Current limitations in TMEM256 antibody research and potential future directions include:

• Limited Functional Characterization:
  The biological function of TMEM256 remains incompletely characterized, making it challenging to develop function-blocking antibodies or assess their biological effects. Future research should further elucidate TMEM256's role in cellular processes, potentially related to its association with extracellular exosomes .

• Species Cross-Reactivity:
  Most available TMEM256 antibodies are primarily reactive with human proteins , with limited validated cross-reactivity to model organisms. Developing antibodies with broader species reactivity would facilitate comparative studies and the use of animal models.

• Application Limitations:
  Current TMEM256 antibodies show variable performance across applications. While many are validated for ELISA, IF, and IHC , fewer options exist for techniques like flow cytometry or chromatin immunoprecipitation. Expanding application versatility would benefit researchers using diverse methodologies.

• Detection Sensitivity Challenges:
  As a low molecular weight membrane protein (11.7 kDa) , TMEM256 may present detection challenges in complex samples. Future development of high-sensitivity detection methods, including signal amplification techniques or proximity ligation approaches, could improve detection limits.

Future directions should include the development of monoclonal antibodies targeting TMEM256, which would provide more consistent lot-to-lot performance compared to the currently predominant polyclonal antibodies . Additionally, antibodies recognizing post-translational modifications of TMEM256 could reveal regulatory mechanisms controlling this protein's function.

How does TMEM256 antibody performance compare across different detection platforms?

TMEM256 antibody performance varies across detection platforms, with important considerations for experimental design:

• Immunohistochemistry vs. Immunofluorescence:
  Several TMEM256 antibodies are validated for both IHC and IF applications , but performance characteristics may differ:

  • IHC typically offers better tissue morphology preservation and long-term sample stability

  • IF provides better spatial resolution and multiplexing capabilities with other antibodies

• ELISA vs. Western Blot:

  • ELISA appears to be more universally supported across TMEM256 antibodies

  • Western blot detection may be challenging due to TMEM256's low molecular weight (11.7 kDa) , potentially requiring specialized gel systems

• Microarray Performance Considerations:
  When using antibody microarrays for TMEM256 detection:

  • At low concentrations of antigen, ratio measurements between replicate spots typically show increased dispersion

  • Signal-to-noise ratios may limit detection, with plateaus occurring as fluorescence signals approach background levels

  • For quantitative analysis, performance is most reliable when antigen concentrations remain above detection thresholds (typically ≥30 ng/ml for many proteins)

• Platform-Specific Optimization:
  Each platform requires specific optimization strategies:

  • For microarrays: Optimization of labeling reactions is critical as this appears to be a more important source of variation than cross-reactivity or dilution errors

  • For tissue-based detection: The performance of TMEM256 antibodies can be significantly influenced by fixation methods and antigen retrieval techniques

When choosing between platforms, researchers should consider that antibodies demonstrating ideal performance in one platform may show non-linear responses in others, necessitating platform-specific validation and optimization.

How can TMEM256 antibodies be incorporated into multiplex detection systems?

TMEM256 antibodies can be integrated into multiplex detection systems through several approaches that allow simultaneous analysis with other proteins of interest:

• Multiplex Immunofluorescence:

  • Combine TMEM256 antibodies with antibodies against other targets using species or isotype differences

  • Use directly conjugated TMEM256 antibodies with different fluorophores (e.g., FITC-conjugated TMEM256 antibodies)

  • Implement sequential detection with antibody stripping between rounds

  • Consider tyramide signal amplification for enhanced sensitivity

• Protein Microarrays:

  • Include TMEM256 antibodies in antibody microarray panels

  • For comparative fluorescence assays, label protein mixtures with distinct fluorophores (e.g., Cy3/Cy5)

  • Analyze log ratios of fluorescence signals to quantify TMEM256 presence across samples

  • Include internal controls to correct for variation in labeling reactions

• Mass Cytometry Approaches:

  • Conjugate TMEM256 antibodies with rare earth metals for CyTOF analysis

  • Combine with dozens of other markers for high-dimensional phenotyping

• Proximity-Based Detection Systems:

  • Pair TMEM256 antibodies with antibodies against potential interaction partners

  • Implement proximity ligation assays or FRET-based approaches to detect protein-protein interactions

When designing multiplex systems, antibody cross-reactivity and compatibility of detection systems are critical considerations. For optimal performance in quantitative multiplex assays, include standard curves with known quantities of recombinant TMEM256 protein.

What role could TMEM256 antibodies play in biomarker development research?

While current literature does not specifically identify TMEM256 as an established biomarker, the available antibodies could support biomarker development research through several approaches:

• Expression Profile Analysis:

  • TMEM256 antibodies can be used to characterize protein expression across tissue types and disease states

  • The availability of antibodies validated for IHC applications facilitates screening of clinical tissue samples

  • Systematic analysis could identify conditions where TMEM256 expression is dysregulated

• Detection Platform Development:

  • The performance characteristics of TMEM256 antibodies in various platforms (ELISA, IF, IHC) provide flexibility for different detection approaches

  • Quantitative assays like ELISA could be developed for measuring TMEM256 levels in patient samples

  • Antibody pairs recognizing different epitopes could be developed for sandwich ELISA applications

• Multi-Marker Panel Integration:

  • TMEM256 detection could be incorporated into multiplexed protein panels

  • Protein microarray approaches allow simultaneous analysis of multiple proteins

  • Statistical analysis could determine if TMEM256 adds diagnostic or prognostic value in combination with other markers

• Technical Validation Requirements:

  • Analytical validation would need to establish sensitivity, specificity, reproducibility, and accuracy

  • Clinical validation would require correlation with disease states or outcomes

  • Harmonization across laboratories would depend on antibody standardization

For advanced biomarker applications, development of monoclonal antibodies against TMEM256 would provide better standardization than the currently predominant polyclonal antibodies .