TMEM217 Antibody

What is TMEM217 and why is it studied in cancer research?

TMEM217 (Transmembrane Protein 217) is a membrane-spanning protein that has garnered research interest due to its potential implications in cancer development and progression pathways. This protein appears to play a significant role in cellular processes related to oncogenic transformation, making it an intriguing target for investigation in cancer biology . Researchers are particularly interested in understanding its biological functions and potential involvement in disease mechanisms, as characterizing its expression patterns and functional properties may contribute to novel therapeutic approaches targeting this protein . The protein has alternative names including C6orf128 and dJ355M6.2, and is encoded by gene ID 221468 .

What validated applications are available for TMEM217 antibodies?

TMEM217 antibodies have been validated for multiple research applications, with specific dilution recommendations for each technique:

These applications enable researchers to detect and quantify TMEM217 expression in various experimental settings, from protein lysates to intact cells . The validated methods provide multiple approaches for investigating TMEM217's role in cellular processes and disease states.

What are the characteristics of currently available TMEM217 antibodies?

The available TMEM217 antibodies exhibit several key characteristics that determine their research applications:

These antibodies have demonstrated high specificity for human TMEM217, making them valuable tools for investigating this protein in human samples and cancer cell lines . The specific binding characteristics enable reliable detection across multiple experimental platforms.

What is the optimal storage and handling protocol for TMEM217 antibodies?

To maintain antibody functionality and extend shelf life, researchers should follow these specific storage and handling guidelines:

• Store antibodies at -20°C or -80°C as specified by the manufacturer

• Prepare small aliquots to avoid repeated freeze-thaw cycles that can degrade antibody quality

• For conjugated antibodies (such as FITC-labeled), avoid exposure to light during handling and storage

• Most TMEM217 antibodies are supplied in a storage buffer containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4)

• When working with FITC-conjugated antibodies, take additional precautions to minimize photobleaching during experimental procedures

Proper storage and handling are essential for maintaining antibody performance and ensuring reproducible experimental results across studies.

How should researchers validate TMEM217 antibody specificity?

Thorough validation of antibody specificity is critical for obtaining reliable research results. For TMEM217 antibodies, researchers should implement a multi-faceted validation approach:

• Western blot verification: Confirm detection of bands at the predicted molecular weights of 27 kDa and 25 kDa in positive control samples such as Jurkat whole cell lysate

• Peptide competition assay: Utilize recombinant TMEM217 protein antigens (such as NBP2-68914PEP) as blocking agents to demonstrate binding specificity

• Cross-application validation: Verify consistent detection patterns across multiple techniques (ELISA, WB, IF) to confirm target specificity

• Knockout/knockdown controls: Where available, include TMEM217-knockout or siRNA-treated samples as negative controls

• Cross-reactivity assessment: Test for potential cross-reactivity with other TMEM family members, especially those with structural similarities

This comprehensive validation strategy ensures that experimental findings genuinely reflect TMEM217 biology rather than non-specific interactions or artifacts.

What are the optimal conditions for immunofluorescence experiments using TMEM217 antibodies?

For successful immunofluorescence detection of TMEM217, researchers should optimize several key parameters:

• Antibody selection: Choose between directly conjugated (e.g., FITC-labeled ABIN6066609) or unconjugated primary antibodies based on experimental design requirements

• Dilution optimization: Start with the recommended dilution range (1:50-1:200) but perform titration experiments to determine optimal concentration for specific cell types

• Fixation method: Test both paraformaldehyde and methanol fixation, as transmembrane protein epitopes may be differentially accessible depending on fixation protocol

• Permeabilization: Use mild detergents (0.1-0.3% Triton X-100 or 0.1% Saponin) to preserve membrane integrity while allowing antibody access

• Blocking strategy: Implement robust blocking (5-10% normal serum from the same species as the secondary antibody) to reduce background

• Controls: Include appropriate negative controls (secondary-only, isotype controls) and positive controls (Jurkat cells)

These optimized conditions enable specific visualization of TMEM217 localization and expression patterns in cellular contexts.

How can researchers troubleshoot Western blot inconsistencies when detecting TMEM217?

When encountering inconsistent Western blot results with TMEM217 antibodies, systematic troubleshooting should address:

• Sample preparation: Ensure complete solubilization of membrane proteins using appropriate detergents (RIPA buffer with 0.1% SDS or NP-40)

• Loading controls: Validate equal loading using membrane protein-specific loading controls rather than cytosolic markers

• Transfer efficiency: Optimize transfer conditions for membrane proteins (longer transfer times, addition of SDS to transfer buffer)

• Antibody concentration: Test multiple dilutions within the recommended range (1:1000-1:5000)

• Blocking optimization: Try different blocking agents (5% milk vs. 5% BSA) to reduce background while preserving specific signal

• Detection system: For challenging samples, consider using high-sensitivity detection systems or signal amplification methods

• Positive control: Always include Jurkat whole cell lysate as a positive control to confirm assay functionality

When blots show multiple bands, researchers should verify whether these represent different isoforms, post-translational modifications, or degradation products of TMEM217.

What experimental considerations are important when quantifying TMEM217 expression levels?

Accurate quantification of TMEM217 expression requires attention to methodological details:

• Western blot quantification:

  • Use gradient gels (4-20%) to improve resolution of membrane proteins

  • Apply rigorous densitometric analysis with appropriate normalization to loading controls

  • Include calibration standards when possible for absolute quantification

• ELISA-based quantification:

  • Optimize antibody pairs for sandwich ELISA development

  • Establish standard curves using recombinant TMEM217 protein

  • Apply recommended dilutions (1:2000-1:10000) but optimize based on sample characteristics

• Immunofluorescence quantification:

  • Implement consistent image acquisition parameters (exposure time, gain)

  • Utilize automated image analysis software with threshold-based quantification

  • Include calibration standards for fluorescence intensity normalization

• Controls and validation:

  • Cross-validate results using multiple quantification methods

  • Include biological replicates to account for natural expression variation

  • Consider the impact of cell confluence and culture conditions on expression levels

This methodical approach ensures reliable quantification of TMEM217 expression across experimental systems.

What approaches can minimize cross-reactivity when working with TMEM217 antibodies?

Cross-reactivity presents a significant challenge when studying transmembrane proteins. To minimize these issues with TMEM217 antibodies:

• Antibody selection: Choose antibodies targeting unique epitopes of TMEM217 that have minimal sequence homology with other TMEM family members

• Pre-absorption: Consider pre-absorbing antibodies with recombinant proteins of closely related TMEM family members to remove cross-reactive antibodies

• Titration optimization: Determine the minimum effective antibody concentration that provides specific signal while minimizing cross-reactivity

• Blocking peptide controls: Use TMEM217-specific blocking peptides like NBP2-68914PEP in parallel experiments to confirm signal specificity

• Alternative detection strategies: Consider using multiple antibodies targeting different epitopes to confirm findings

• Stringent washing: Implement more stringent washing conditions to remove weakly bound, potentially cross-reactive antibodies

These approaches help ensure that observed signals genuinely represent TMEM217 rather than related transmembrane proteins.

How should controls be integrated in TMEM217 antibody-based experiments?

A robust control strategy is essential for generating reliable data with TMEM217 antibodies:

This comprehensive control framework enables confident interpretation of experimental results and facilitates troubleshooting when unexpected results occur.

What are the considerations for multi-color immunofluorescence involving TMEM217?

When designing multi-color immunofluorescence experiments that include TMEM217 detection:

• Antibody host species: Select TMEM217 antibodies and other target antibodies from different host species to enable clear secondary antibody discrimination

• Fluorophore selection: Choose fluorophores with minimal spectral overlap, particularly when using FITC-conjugated TMEM217 antibodies

• Sequential staining: Consider sequential rather than simultaneous staining protocols for challenging combinations

• Cross-blocking: Implement additional blocking steps between primary antibody incubations to prevent cross-reactivity

• Controls: Include single-color controls to establish proper compensation and identify potential bleed-through

• Fixation compatibility: Ensure all antibodies in the panel perform optimally under the same fixation conditions

• Order of application: Optimize the sequence of antibody application, typically applying the weakest signal antibody first

These considerations enable simultaneous visualization of TMEM217 alongside other proteins of interest, facilitating colocalization studies and pathway analyses.

What are the critical factors in selecting the appropriate TMEM217 antibody format for specific research applications?

The selection of the optimal TMEM217 antibody format should be guided by application-specific requirements:

When selecting antibodies, researchers should also consider the epitope location (e.g., ABIN6066609 targets AA 150-211) , as this impacts accessibility in different applications and experimental conditions.

What is the recommended protocol for Western blot detection of TMEM217?

For optimal Western blot detection of TMEM217, researchers should follow this specialized protocol:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors for cell lysis

  • Heat samples at 70°C (not 95°C) for 10 minutes to prevent membrane protein aggregation

• Gel electrophoresis:

  • Load 20-50 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 27 kDa and 25 kDa TMEM217 bands

• Transfer:

  • Perform wet transfer at 30V overnight at 4°C for efficient transfer of membrane proteins

  • Use PVDF membrane (0.45 μm) pre-activated with methanol

• Antibody incubation:

  • Block with 5% BSA in TBST (preferred over milk for membrane proteins)

  • Incubate with TMEM217 antibody at 1:1000 to 1:5000 dilution overnight at 4°C

  • Use goat anti-rabbit IgG-HRP at 1:10000 as secondary antibody

• Detection:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expect bands at approximately 27 kDa and 25 kDa

This protocol has been optimized for the detection of TMEM217 in Jurkat whole cell lysates but may require adjustment for other sample types.

How can researchers optimize immunofluorescence detection of TMEM217 in different cell types?

Optimizing immunofluorescence detection of TMEM217 across diverse cell types requires systematic adjustment of several parameters:

• Fixation method comparison:

  • Test 4% paraformaldehyde (10 minutes) versus methanol (5 minutes at -20°C)

  • Evaluate which method best preserves TMEM217 epitopes while maintaining cellular architecture

• Permeabilization optimization:

  • Compare mild (0.1% Triton X-100, 5 minutes) versus moderate (0.3% Triton X-100, 10 minutes) permeabilization

  • For challenging samples, test saponin-based permeabilization to better preserve membrane proteins

• Antibody dilution matrix:

  • Test a range of dilutions from 1:50 to 1:200 for primary antibody

  • Optimize secondary antibody concentration independently

• Signal amplification:

  • For low-expressing cell types, consider tyramide signal amplification or higher sensitivity detection systems

  • Evaluate direct detection (FITC-conjugated antibody) versus indirect detection with amplification

• Incubation conditions:

  • Compare room temperature (1 hour) versus 4°C (overnight) primary antibody incubation

  • Assess the impact of adding protein carriers (0.1-1% BSA) to antibody diluent

This systematic optimization approach enables reliable TMEM217 detection across diverse experimental systems.

What approaches are effective for troubleshooting non-specific binding with TMEM217 antibodies?

When faced with non-specific binding issues when using TMEM217 antibodies, researchers should implement this hierarchical troubleshooting approach:

• Blocking optimization:

  • Test alternative blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (1-2 hours) and concentration (3-5%)

• Antibody concentration adjustment:

  • Create a dilution series spanning and extending beyond the recommended range

  • Identify the optimal concentration that maximizes specific signal while minimizing background

• Washing stringency:

  • Increase wash buffer stringency (0.1% to 0.3% Tween-20)

  • Extend washing times and increase the number of wash steps

• Pre-absorption strategy:

  • Pre-absorb the antibody with the immunizing peptide or recombinant TMEM217 protein

  • Include control experiments using the pre-absorbed antibody to identify non-specific signals

• Alternative antibody evaluation:

  • Test multiple antibodies targeting different epitopes of TMEM217

  • Compare monoclonal versus polyclonal antibody performance

• Sample preparation reassessment:

  • Evaluate whether sample preparation methods might be exposing epitopes that promote non-specific binding

  • Test alternative fixation methods that might reduce non-specific interactions

This systematic approach helps identify and address the specific source of non-specific binding issues.

How can TMEM217 antibodies be utilized to investigate its role in cancer progression?

TMEM217 antibodies enable multiple investigative approaches to elucidate this protein's role in cancer:

• Expression profiling:

  • Use TMEM217 antibodies for immunohistochemistry on tissue microarrays to assess expression across cancer types and stages

  • Correlate expression patterns with clinical outcomes and other molecular markers

• Functional studies:

  • Combine TMEM217 antibodies with cell proliferation, migration, and invasion assays to correlate expression with cancer hallmarks

  • Use blocking antibodies to investigate whether TMEM217 function can be inhibited externally

• Signaling pathway analysis:

  • Apply TMEM217 antibodies in co-immunoprecipitation studies to identify binding partners

  • Employ multi-color immunofluorescence to assess colocalization with known oncogenic pathway components

• Diagnostic potential assessment:

  • Evaluate TMEM217 antibodies for potential diagnostic applications in cancer detection

  • Determine sensitivity and specificity in distinguishing cancerous from normal tissues

These approaches leverage TMEM217 antibodies as tools to understand its role in cancer progression, potentially leading to novel therapeutic strategies targeting this protein .

What methodological advances might improve TMEM217 antibody performance in challenging applications?

Emerging methodological advances that could enhance TMEM217 antibody performance include:

• Single-domain antibody development:

  • Smaller antibody formats may access epitopes in transmembrane proteins that conventional antibodies cannot reach

  • Camelid-derived nanobodies or synthetic single-domain antibodies might offer improved specificity

• Native conformation preservation:

  • Developing sample preparation methods that better preserve membrane protein native conformation

  • Utilizing mild detergents and alternative solubilization approaches for improved epitope accessibility

• Proximity-based detection systems:

  • Integrating TMEM217 antibodies with proximity ligation assays for improved sensitivity

  • Applying CRISPR-based epitope tagging to enable alternative detection strategies

• Computational epitope analysis:

  • Using advanced structural prediction algorithms to identify optimal epitopes for antibody development

  • Designing antibodies against highly specific regions with minimal homology to other TMEM proteins

These methodological advances could significantly improve the specificity, sensitivity, and versatility of TMEM217 antibodies for research applications.