TMEM214 Antibody, Biotin conjugated

What is TMEM214 and why is it studied in research?

Transmembrane protein 214 (TMEM214) is a 77 kDa membrane protein that is widely expressed at high levels in various tissues. The gene encoding this protein maps to chromosome 2, specifically at position 2p23.3. TMEM214 serves as a critical mediator in endoplasmic reticulum stress-induced apoptosis, operating in cooperation with CASP4. It is required for the activation of CASP4 following endoplasmic reticulum stress, making it an important target for studies involving cellular stress responses and programmed cell death pathways . Research involving TMEM214 often focuses on cellular biology, particularly mechanisms related to endoplasmic reticulum function and cellular survival.

What are the key specifications of commercially available biotin-conjugated TMEM214 antibodies?

Biotin-conjugated TMEM214 antibodies are typically polyclonal antibodies raised in rabbits. The most common specifications include:

• Target binding region: AA 2-236 of the human TMEM214 protein

• Host species: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Purification method: Protein G purification (>95% purity)

• Immunogen: Recombinant Human Transmembrane protein 214 protein (amino acids 2-236)

• Primary application: ELISA

• Reactivity: Human (primary), with some products showing cross-reactivity with mouse and rat

How does biotin conjugation affect the application of TMEM214 antibodies?

Biotin conjugation provides several advantages for TMEM214 antibody applications. The strong affinity between biotin and streptavidin/avidin allows for enhanced detection sensitivity in various assays. This conjugation enables versatile detection methods through secondary reagents like streptavidin-HRP, streptavidin-fluorophores, or other avidin-conjugated detection systems without requiring species-specific secondary antibodies. The biotin tag also allows for signal amplification strategies that can significantly improve detection in samples with low TMEM214 expression. Additionally, biotin conjugation facilitates multiplex experiments where several antibodies may be used simultaneously, as the biotin-streptavidin detection system can be combined with other detection methods .

What are the recommended storage conditions for biotin-conjugated TMEM214 antibodies?

For optimal stability and performance, biotin-conjugated TMEM214 antibodies should be stored at -20°C for long-term preservation. Most commercial preparations remain stable for one year when properly stored. The antibodies are typically supplied in buffers containing preservatives like sodium azide (0.02-0.09%) and stabilizers such as glycerol (often at 50% concentration) with PBS at pH 7.3-7.4. It is advisable to avoid repeated freeze-thaw cycles, which can degrade antibody performance. For antibodies supplied in larger volumes, aliquoting before freezing is recommended, though some suppliers indicate that aliquoting is unnecessary for -20°C storage of certain preparations . Thawed antibodies should be kept at 4°C if they will be used within three months .

How do different epitope targets within TMEM214 affect antibody performance across applications?

Different commercial TMEM214 antibodies target various epitope regions, which significantly impacts their performance across applications:

What are the critical considerations when optimizing biotin-conjugated TMEM214 antibodies for immunofluorescence studies?

When optimizing biotin-conjugated TMEM214 antibodies for immunofluorescence:

• Dilution optimization: Starting dilutions typically range from 1:25 to 1:100 for IF applications, but must be empirically determined for each experimental system. Titration experiments are essential for determining optimal signal-to-noise ratio .

• Fixation method selection: Different fixation protocols (paraformaldehyde, methanol, or acetone) may affect epitope accessibility. For membrane proteins like TMEM214, paraformaldehyde fixation often preserves membrane structure better.

• Permeabilization considerations: Since TMEM214 is a transmembrane protein, the permeabilization step is critical. Detergents like Triton X-100 (0.1-0.5%) or saponin (0.1%) may be used, with optimization necessary to balance accessibility with epitope preservation.

• Signal amplification: The biotin-streptavidin system allows for amplification using streptavidin conjugated to bright fluorophores. Multiple amplification systems may be used if signal strength is inadequate.

• Blocking optimization: Thorough blocking is essential to prevent non-specific binding of both the biotinylated antibody and the streptavidin detection reagent. BSA (1-5%) or normal serum (5-10%) from a species different from the host is recommended.

• Endogenous biotin blocking: Tissues with high endogenous biotin (like liver, kidney, brain) require additional blocking steps with avidin/biotin blocking kits to reduce background .

Published applications have successfully demonstrated TMEM214 localization in human muscle slides using properly optimized immunofluorescence protocols .

How can researchers confirm the specificity of TMEM214 antibody binding in experimental systems?

Confirming specificity of TMEM214 antibody binding requires multiple validation approaches:

• Western blot validation: Confirm detection of a single band at the expected molecular weight of 77 kDa. The observed molecular weight for TMEM214 aligns well with the calculated weight (77.2 kDa), making this a reliable validation approach .

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR-Cas9 knockout of TMEM214 and demonstrate reduced or absent antibody signal compared to controls.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide prior to the primary application. Signal reduction indicates specific binding to the target epitope .

• Multiple antibody validation: Test different antibodies targeting distinct TMEM214 epitopes (e.g., comparing antibodies targeting AA 2-236 versus AA 38-66) to confirm consistent localization or detection patterns .

• Cross-species validation: For antibodies with cross-reactivity to multiple species, confirm similar detection patterns across species, accounting for known biological variations .

• Positive control tissues/cells: Use samples known to express TMEM214 at high levels, such as C2C12 cells, which have been validated in Western blot applications with TMEM214 antibodies .

What are the implications of TMEM214's role in endoplasmic reticulum stress-induced apoptosis for experimental design?

TMEM214's critical role as a mediator of endoplasmic reticulum (ER) stress-induced apoptosis, particularly its cooperation with CASP4, creates several important considerations for experimental design:

What are the optimal protocols for using biotin-conjugated TMEM214 antibodies in ELISA applications?

For optimal ELISA applications with biotin-conjugated TMEM214 antibodies:

• Coating conditions: When developing a sandwich ELISA, coat plates with capture antibody (anti-TMEM214 targeting a different epitope) at 1-2 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C.

• Sample preparation: For cell lysates, use a non-denaturing lysis buffer containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), and protease inhibitors. For tissue samples, homogenization in PBS with protease inhibitors followed by centrifugation is recommended.

• Biotin-conjugated antibody dilution: Based on manufacturer recommendations, use biotin-conjugated TMEM214 antibodies at dilutions ranging from 1:1000 to 1:5000, optimizing through titration experiments for each specific lot and application.

• Detection system: Utilize streptavidin-HRP at 1:5000 to 1:20000 dilution, followed by TMB substrate development for colorimetric detection.

• Blocking conditions: Block with 2-5% BSA or 5% non-fat dry milk in PBS-T (PBS with 0.05% Tween-20) for 1-2 hours at room temperature to minimize background.

• Washing stringency: Perform 4-5 washes with PBS-T between each step, with the final wash being more extensive (5-6 times) to reduce background signal.

• Controls: Include recombinant TMEM214 protein standards (covering the AA 2-236 region) for quantitative analysis, along with negative controls lacking the target antigen .

How should researchers handle potential cross-reactivity when using TMEM214 antibodies across different species?

Managing cross-reactivity when using TMEM214 antibodies across species requires systematic validation approaches:

• Sequence homology analysis: Before experimental application, compare TMEM214 protein sequences across target species, particularly focusing on the epitope region. For example, antibodies targeting AA 2-236 or AA 70-120 have demonstrated cross-reactivity with human, mouse, and rat TMEM214 .

• Validation in each species: When using an antibody in a new species, perform Western blot validation to confirm the detected protein matches the expected molecular weight (approximately 77 kDa for TMEM214). Note that slight variations in molecular weight may occur between species due to post-translational modifications.

• Positive control selection: Use well-characterized samples known to express TMEM214 in each species, such as C2C12 cells for mouse studies, which have been validated in Western blot applications .

• Dilution optimization by species: When cross-reactivity is confirmed, optimal working dilutions may vary by species. For example:

  • For human samples: WB 1:500-1:1000, IF 1:25-1:100

  • For mouse/rat samples: May require adjusted dilutions based on epitope conservation

• Non-specific binding controls: Include samples from TMEM214 knockout models or tissues known not to express TMEM214 as negative controls to assess non-specific binding in each species.

• Publication verification: Reference published applications demonstrating successful use of specific TMEM214 antibodies in your target species. For instance, published studies have validated specific antibodies for immunohistochemistry applications in human muscle samples .

What troubleshooting approaches are recommended when working with biotin-conjugated TMEM214 antibodies?

When troubleshooting issues with biotin-conjugated TMEM214 antibodies:

For persistent issues, researchers should contact manufacturers for lot-specific recommendations, as biotin conjugation efficiency can vary between production lots .

What considerations are important when using TMEM214 antibodies in the context of Emery-Dreifuss muscular dystrophy research?

When using TMEM214 antibodies in Emery-Dreifuss muscular dystrophy (EDMD) research, several specialized considerations are important:

• Nuclear envelope localization assessment: Published research indicates that nuclear envelope proteins may serve as inconsistent markers for EDMD pathology. When evaluating TMEM214 distribution, compare against other established nuclear envelope markers to identify disease-specific changes versus general nuclear envelope perturbations .

• Tissue sample preparation optimization: For muscular dystrophy samples, optimal fixation methods are critical. Paraformaldehyde fixation (4%, 10-15 minutes) typically preserves both membrane structures and nuclear architecture needed for proper TMEM214 evaluation.

• Immunofluorescence protocols for muscle tissue: For EDMD muscle slides, the recommended dilution range for TMEM214 antibodies is 1:25-1:100, with additional optimization suggested for each specific sample type. Muscle tissue may require extended permeabilization steps compared to cultured cells .

• Comparative analysis approach: Always analyze EDMD samples alongside matched healthy controls using identical protocols. This is particularly important given the reported inconsistency of nuclear envelope protein markers in EDMD pathology evaluation .

• Quantitative assessment methods: Implement quantitative analysis of TMEM214 staining patterns, including intensity measurements and distribution profiling (particularly nuclear envelope to cytoplasmic ratio) to detect subtle changes in EDMD samples.

• Multi-protein co-localization studies: Combine TMEM214 antibodies with markers for other relevant proteins (e.g., emerin, lamin A/C, lamin B) in co-localization studies to better characterize EDMD-specific changes in nuclear envelope organization .

What are the most effective protein extraction methods when preparing samples for TMEM214 antibody applications?

Optimizing protein extraction for TMEM214 analysis requires considering its transmembrane nature and subcellular localization:

• Membrane protein extraction buffers: Use buffers containing mild detergents that efficiently solubilize membrane proteins while preserving epitope structure:

  • For Western blot: RIPA buffer (150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) supplemented with protease inhibitors

  • For immunoprecipitation: Milder buffers (1% NP-40 or 1% Digitonin in PBS with protease inhibitors) better preserve protein-protein interactions

• Subcellular fractionation approach: For focused analysis of TMEM214 in its native compartments:

  • Separate nuclear, cytoplasmic, and membrane fractions using sequential extraction

  • Enrich for endoplasmic reticulum fraction using sucrose gradient ultracentrifugation when specifically studying ER stress responses

• Sample heating considerations: Limited heat exposure (70°C for 5 minutes instead of 95°C boiling) may better preserve transmembrane protein structure when preparing for SDS-PAGE.

• Protein concentration determination: Use detergent-compatible protein assays (BCA or modified Lowry) that account for the presence of membrane-extraction detergents.

• Loading controls selection: When analyzing TMEM214 by Western blot, use appropriate loading controls for membrane proteins (such as Na⁺/K⁺-ATPase or calnexin) rather than general cytoskeletal markers .

The observed molecular weight of TMEM214 in properly prepared samples should be approximately 77 kDa across human, mouse, and rat samples, consistent with the calculated molecular weight of 77.2 kDa .

How can researchers optimize Western blot protocols specifically for biotin-conjugated TMEM214 antibodies?

Optimizing Western blot protocols for biotin-conjugated TMEM214 antibodies requires specific adjustments:

• Sample preparation: Use membrane protein-optimized lysis buffers with complete protease inhibitors. For transmembrane proteins like TMEM214, avoid extended boiling; instead, heat samples at 70°C for 5-10 minutes to reduce aggregation.

• Gel percentage selection: Use 8-10% polyacrylamide gels for optimal resolution around the expected 77 kDa molecular weight of TMEM214.

• Transfer optimization: Employ wet transfer systems with 20% methanol buffer for efficient transfer of this membrane protein. For TMEM214, extended transfer times (90-120 minutes) or semi-dry systems with modified buffers may improve transfer efficiency.

• Blocking considerations: When using biotin-conjugated antibodies, standard milk-based blocking solutions may contain endogenous biotin that interferes with detection. Instead, use:

  • 3-5% BSA in TBS-T (preferred) or

  • Commercial biotin-free blocking reagents

• Primary antibody incubation: Based on manufacturer recommendations, use biotin-conjugated TMEM214 antibodies at dilutions of 1:500-1:1000 in blocking buffer, incubating overnight at 4°C for optimal results .

• Detection system: Employ streptavidin-HRP (1:5000-1:10000) rather than secondary antibodies, incubating for 30-60 minutes at room temperature.

• Signal development optimization: For TMEM214 detection, enhanced chemiluminescence (ECL) substrates with moderate to high sensitivity are typically sufficient. For low expression samples, consider using higher sensitivity ECL or alternative detection methods.

• Positive control inclusion: Include C2C12 cell lysate as a positive control, which has been validated to express detectable levels of TMEM214 .

What quantitative methods can be used to analyze TMEM214 expression levels using antibody-based techniques?

Researchers can employ several quantitative methods to analyze TMEM214 expression:

• Quantitative Western blotting:

  • Use internal loading controls appropriate for membrane proteins (Na⁺/K⁺-ATPase, calnexin)

  • Establish standard curves using recombinant TMEM214 protein

  • Employ digital imaging systems with linear dynamic range for densitometric analysis

  • Calculate relative expression using software like ImageJ or dedicated Western blot quantification programs

• Quantitative ELISA approaches:

  • Develop sandwich ELISA using capture antibodies targeting one TMEM214 epitope and biotin-conjugated detection antibodies targeting another

  • Establish standard curves using recombinant TMEM214 (AA 2-236) at concentrations of 0-1000 ng/ml

  • Analyze using 4-parameter logistic regression for accurate concentration determination

• Flow cytometry quantification:

  • Use calibration beads with known antibody binding capacity

  • Calculate molecules of equivalent soluble fluorochrome (MESF) or antibody binding capacity (ABC)

  • Apply biotin-conjugated TMEM214 antibodies at 1:10-1:50 dilutions followed by streptavidin-fluorophore detection

  • Quantify using median fluorescence intensity relative to calibration standards

• Quantitative immunofluorescence:

  • Include calibration standards in each experiment

  • Employ consistent image acquisition parameters

  • Use software for automated quantification of signal intensity and subcellular distribution

  • Apply specialized analysis for membrane protein distribution patterns

• High-content imaging analysis:

  • Combine immunofluorescence with automated microscopy

  • Quantify parameters like TMEM214 intensity, subcellular localization, and co-localization with other markers

  • Analyze large cell populations for statistically robust measurements

When reporting quantitative results, always include details about normalization methods, statistical approaches, and technical replicates to ensure reproducibility .

How might advances in antibody technology impact future research applications of TMEM214 antibodies?

Emerging antibody technologies will likely enhance TMEM214 research capabilities in several important ways:

• Single-domain antibodies and nanobodies: The development of smaller antibody formats may improve access to sterically hindered epitopes in the transmembrane regions of TMEM214, potentially revealing functional domains previously difficult to study with conventional antibodies.

• Site-specific biotin conjugation: Advanced biotin conjugation methods that target specific residues rather than random lysines could improve consistency between antibody lots and preserve epitope binding regions, enhancing reproducibility in TMEM214 detection assays.

• Multiparametric antibody panels: Development of highly compatible TMEM214 antibody panels with minimal cross-reactivity will facilitate simultaneous detection of TMEM214 alongside interaction partners like CASP4, enabling more comprehensive analysis of stress response pathways.

• Intrabodies and live-cell compatible formats: Modified antibody formats designed for intracellular expression or membrane permeability could enable live-cell imaging of TMEM214 dynamics during ER stress responses, providing temporal resolution currently unavailable.

• Proximity labeling antibody conjugates: Conjugation of TMEM214 antibodies with enzymes like APEX2 or TurboID could enable spatial proteomics applications, identifying novel proteins in proximity to TMEM214 under various cellular conditions.

• Automated validation pipelines: Implementation of high-throughput antibody validation systems will provide more comprehensive cross-reactivity profiles and application-specific performance metrics, increasing confidence in experimental outcomes using TMEM214 antibodies .

These technological advances will likely expand the utility of TMEM214 antibodies beyond current applications in fundamental research to potentially include diagnostic applications and therapeutic development targeting ER stress pathways.

What are the emerging research areas where TMEM214 antibodies may provide valuable insights?

TMEM214 antibodies are poised to contribute to several emerging research areas:

• ER stress in neurodegenerative diseases: Given TMEM214's role in ER stress-induced apoptosis, antibodies targeting this protein may provide new insights into neurodegenerative conditions where ER stress is implicated, including Alzheimer's, Parkinson's, and ALS. Quantitative analysis of TMEM214 expression and localization in disease models could reveal novel therapeutic targets.

• Cancer therapy resistance mechanisms: ER stress response pathways contribute to therapy resistance in multiple cancer types. TMEM214 antibodies may help characterize how cancer cells modulate these pathways to survive treatment, potentially identifying biomarkers for treatment response.

• Muscular dystrophy pathophysiology: Building on published applications in Emery-Dreifuss muscular dystrophy, TMEM214 antibodies could help clarify the relationship between nuclear envelope disruption and disease progression across muscular dystrophy subtypes .

• Cellular adaptation to metabolic stress: As cellular metabolism and ER function are tightly linked, TMEM214 antibodies may help elucidate how metabolic perturbations affect ER stress responses in conditions like diabetes and metabolic syndrome.

• Developmental biology: TMEM214's role during cellular differentiation and tissue development remains largely unexplored. Antibodies could reveal dynamic expression patterns during embryonic development and tissue specialization.

• Single-cell analysis of heterogeneous responses: Combining TMEM214 antibodies with single-cell technologies may reveal previously unappreciated heterogeneity in how individual cells within a population respond to ER stress challenges, potentially explaining variable outcomes in disease settings.

• Extracellular vesicle research: Investigating whether TMEM214 or its fragments are incorporated into exosomes or other extracellular vesicles during cellular stress could open new avenues for biomarker development .