TMEM214 Antibody, HRP conjugated

What is TMEM214 and what is its primary biological function?

TMEM214 (Transmembrane protein 214) is a critical mediator of endoplasmic reticulum (ER) stress-induced apoptosis. The protein is localized on the outer membrane of the ER and constitutively associates with procaspase 4, which is essential for ER stress-induced apoptosis . TMEM214 contains two transmembrane domains at its C-terminus and a large N-terminal domain that extends into the cytosol . The N-terminal cytoplasmic region (amino acids 176-354) is required for its interaction with procaspase 4, while either of its two transmembrane domains (amino acids 480-500 or 616-636) is sufficient for its localization to the ER . Functionally, TMEM214 anchors procaspase 4 to the ER, facilitating its activation following ER stress, which subsequently triggers the apoptotic cascade .

How is TMEM214 involved in endoplasmic reticulum stress pathways?

TMEM214 plays a specific role in ER stress-induced apoptosis but appears to be independent of the unfolded protein response (UPR) pathway. Studies have shown that overexpression of TMEM214 does not significantly affect protein or mRNA levels of UPR markers like GRP78, GRP94, and PDI . Instead, TMEM214 mediates apoptosis through a distinct mechanism involving caspase 4. When cells experience ER stress induced by agents such as thapsigargin (TG) or brefeldin A (BFA), TMEM214 facilitates the activation of procaspase 4, leading to apoptotic cell death . Notably, knockdown of TMEM214 significantly inhibits apoptosis triggered by ER stress inducers but has minimal effect on apoptosis initiated by external stimuli like TNFα or mitochondrion-dependent apoptotic inducers such as actinomycin D and etoposide .

What are the key specifications of TMEM214 Antibody, HRP conjugated?

The TMEM214 Antibody, HRP conjugated (Product Code: CSB-PA023808LB01HU) is a polyclonal antibody raised in rabbits against recombinant Human Transmembrane protein 214 (amino acids 2-236) . This antibody is specifically designed for research applications and has the following specifications:

This antibody is provided for research use only and not intended for diagnostic or therapeutic applications .

What applications is the TMEM214 Antibody, HRP conjugated validated for?

• ELISA: Direct detection of TMEM214 in human samples

• Immunohistochemistry: Potential application for tissue sections with proper optimization

• Western blot: Direct detection capability with chemiluminescent or colorimetric substrates

When considering applications beyond ELISA, researchers should perform thorough validation experiments to confirm antibody performance in their specific experimental system .

What are the optimal storage and handling conditions for TMEM214 Antibody, HRP conjugated?

For optimal performance and longevity of the TMEM214 Antibody, HRP conjugated, adherence to proper storage and handling protocols is essential:

• Storage temperature: Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles: These can degrade both the antibody protein and the HRP enzyme activity

• For working aliquots: Store small volumes at 2-8°C for short-term use (up to 1 month)

• Buffer considerations: The antibody is provided in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• Handling precautions: Always use clean pipette tips and sterile tubes when handling the antibody to prevent contamination

• Light sensitivity: As an HRP-conjugated product, minimize exposure to direct light during handling to preserve enzymatic activity

The manufacturer indicates that proper storage should maintain antibody efficacy for approximately one year from the date of receipt .

What controls should be included when using TMEM214 Antibody in experimental workflows?

Rigorous experimental design requires appropriate controls to ensure valid interpretation of results when using TMEM214 Antibody:

• Positive controls:

  • Cell lines known to express TMEM214 (e.g., HeLa, HCT116, HepG2, and A549 cell lines have documented TMEM214 expression)

  • Recombinant TMEM214 protein

  • Tissues with confirmed TMEM214 expression

• Negative controls:

  • TMEM214 knockdown cells using validated RNAi or CRISPR approaches

  • Cell lines with minimal TMEM214 expression

  • Isotype control (rabbit IgG) to assess non-specific binding

• Procedural controls:

  • Omission of primary antibody

  • Competitive blocking with immunogen peptide

  • Gradient dilution series to determine optimal antibody concentration

• Validation controls:

  • Using multiple antibodies targeting different epitopes of TMEM214

  • Correlation of protein detection with mRNA expression data

These controls are particularly important when investigating the role of TMEM214 in ER stress-induced apoptosis pathways, where specificity of detection is crucial for accurate interpretation of results .

How can TMEM214 Antibody be used to investigate the role of TMEM214 in ER stress-induced apoptosis?

Investigating the role of TMEM214 in ER stress-induced apoptosis requires a multifaceted approach, with the TMEM214 antibody serving as a critical tool:

• Co-localization studies:

  • Use TMEM214 antibody alongside ER markers to confirm localization at the ER membrane

  • Perform co-immunoprecipitation experiments to detect the TMEM214-procaspase 4 complex that forms constitutively, regardless of ER stress induction

• Stress response analysis:

  • Monitor TMEM214 localization and levels before and after ER stress induction using agents such as thapsigargin (TG) or brefeldin A (BFA)

  • Compare results across different cell types, as the correlation between TMEM214 levels and sensitivity to TG-induced apoptosis varies among cell lines (HeLa, HCT116, HepG2, and A549)

• Functional studies:

  • Use TMEM214 antibody to detect changes in protein levels and localization in knockdown/overexpression experiments

  • Monitor procaspase 4 recruitment to the ER in TMEM214 knockdown cells versus control cells to validate TMEM214's role as an anchoring protein

  • Track caspase 4 activation and PARP-1 cleavage as downstream indicators of the TMEM214-mediated apoptotic pathway

• Pathway discrimination:

  • Use TMEM214 antibody alongside markers for other ER stress pathways (CHOP, JNK phosphorylation) to distinguish between different mechanisms of ER stress-induced apoptosis

This approach allows researchers to comprehensively investigate TMEM214's specific role in ER stress-induced apoptosis, distinct from other apoptotic pathways.

What methodological considerations are important when studying TMEM214-procaspase 4 interactions?

The TMEM214-procaspase 4 interaction is central to understanding ER stress-induced apoptosis mechanisms. When investigating this interaction, several methodological considerations are critical:

• Interaction detection methods:

  • Co-immunoprecipitation (Co-IP): Use anti-TMEM214 antibody to pull down the protein complex and then probe for procaspase 4, or vice versa

  • Proximity ligation assay (PLA): Visualize the interaction in situ within cells

  • FRET/BRET assays: For real-time monitoring of protein-protein interactions

• Domain mapping considerations:

  • The N-terminal cytoplasmic region of TMEM214 (amino acids 176-354) is required for procaspase 4 binding

  • Use of truncation mutants can help define the minimal binding domain with greater precision

• Functional validation:

  • Mutational analysis of the binding interface

  • Use of dominant-negative procaspase 4(C284S) to block TMEM214-induced apoptosis

  • Monitor the effect of TMEM214 knockdown on procaspase 4 recruitment to the ER using subcellular fractionation

• Temporal dynamics:

  • While the interaction is constitutive (present with or without ER stress stimulation), the activation mechanism during ER stress remains to be fully elucidated

  • Time-course experiments following ER stress induction can provide insights into any conformational changes or post-translational modifications

Understanding these methodological considerations is essential for designing experiments that accurately characterize the TMEM214-procaspase 4 interaction and its role in apoptotic signaling.

What are common troubleshooting steps when TMEM214 Antibody yields inconsistent results?

When researchers encounter inconsistent results with TMEM214 Antibody, HRP conjugated, several troubleshooting steps should be considered:

• Antibody quality assessment:

  • Check antibody expiration date and storage conditions

  • Validate antibody performance using positive control samples

  • Consider testing antibody lot-to-lot variation if inconsistencies persist

• Protocol optimization:

  • Titrate antibody concentration to determine optimal working dilution (recommended starting range: 1:500-1:1000 for WB applications)

  • Adjust incubation times and temperatures

  • Modify blocking solutions to reduce background (BSA vs. milk-based blockers)

  • For HRP-conjugated antibodies, ensure substrate freshness and proper development time

• Sample-specific issues:

  • Ensure proper protein extraction methods for membrane proteins like TMEM214

  • Use fresh samples or properly stored lysates/tissues

  • Consider the impact of different detergents for membrane protein solubilization

  • Validate that your experimental conditions do not affect TMEM214 protein levels

• Detection system troubleshooting:

  • For HRP-conjugated antibodies, check substrate quality and detection system functionality

  • Ensure proper exposure times when using chemiluminescent detection methods

  • Consider using enhanced detection reagents for low-abundance targets

• Technical verification:

  • Perform parallel experiments using different TMEM214 antibodies targeting distinct epitopes

  • Compare results with orthogonal methods (e.g., mRNA expression analysis)

  • Consider using recombinant TMEM214 as a positive control standard

Systematic application of these troubleshooting steps can help identify and resolve sources of inconsistency in TMEM214 detection.

How can TMEM214 Antibody protocols be optimized for different cell types and tissues?

Optimization of TMEM214 Antibody protocols for various cell types and tissues requires consideration of several factors:

• Expression level adjustments:

  • Different cell lines show variable TMEM214 expression levels, potentially requiring adjusted antibody dilutions

  • HeLa, HCT116, HepG2, and A549 cells have documented TMEM214 expression but at varying levels

  • Consider using Western blot to establish baseline expression in your model system before proceeding to other applications

• Cell/tissue-specific optimization:

  • For adherent cell lines: Standard fixation protocols (4% paraformaldehyde) are generally suitable

  • For suspension cells: Consider cytospin preparation before fixation for immunofluorescence

  • For tissues: Optimize antigen retrieval methods (heat-induced vs. enzymatic) based on tissue type

  • For muscle tissues: Special considerations may be needed as TMEM214 has been examined in muscle slides

• Application-specific modifications:

  • For immunofluorescence: Recommended dilution ranges from 1:25-1:100

  • For Western blot: Recommended dilution ranges from 1:500-1:1000

  • For ELISA: Follow manufacturer's specific recommendations for the HRP-conjugated format

• Buffer and reagent modifications:

  • Consider membrane protein-specific lysis buffers containing appropriate detergents

  • For fixed tissues, extend antibody incubation times to ensure adequate penetration

  • Adjust blocking reagents based on tissue type (BSA vs. serum vs. commercial blockers)

• Signal enhancement strategies:

  • For low-expressing samples, consider using amplification systems compatible with HRP

  • Extend substrate incubation time for weak signals, being careful to monitor background

  • Use of tyramide signal amplification (TSA) for trace amounts of target protein

Each cell type and tissue may require specific protocol adjustments to achieve optimal TMEM214 detection while maintaining specificity and signal-to-noise ratio.

How can TMEM214 Antibody be used to study ER stress pathways in disease models?

TMEM214 Antibody can be a valuable tool for investigating ER stress pathways in various disease models, particularly those involving dysfunctional apoptotic responses:

• Neurodegenerative diseases:

  • ER stress is implicated in Alzheimer's, Parkinson's, and ALS pathogenesis

  • TMEM214 antibody can help assess the activation of the caspase 4-dependent apoptotic pathway in neuronal models

  • Correlation of TMEM214-procaspase 4 interaction with disease progression may identify novel therapeutic targets

• Cancer research applications:

  • Many cancer cells develop resistance to ER stress-induced apoptosis

  • TMEM214 antibody can help determine whether altered TMEM214 expression or localization contributes to this resistance

  • Comparison between cancer and normal tissues may reveal cancer-specific alterations in TMEM214-mediated apoptosis

• Inflammatory conditions:

  • ER stress plays a role in inflammatory bowel disease and rheumatoid arthritis

  • TMEM214 antibody can help assess whether the TMEM214-caspase 4 axis contributes to inflammation-associated tissue damage

  • Evaluation of TMEM214 as a potential biomarker for disease activity

• Metabolic disorders:

  • ER stress is a key feature in obesity, diabetes, and fatty liver disease

  • TMEM214 antibody can help determine whether metabolic stress affects TMEM214-mediated apoptosis in pancreatic β-cells or hepatocytes

  • Correlation of TMEM214 levels with disease severity may provide prognostic insights

• Methodological approaches:

  • Immunohistochemistry on patient samples to assess TMEM214 expression and localization

  • Co-localization studies with ER stress markers and apoptotic indicators

  • Comparison of TMEM214-procaspase 4 complex formation in disease vs. healthy controls

These applications can provide valuable insights into the role of TMEM214-mediated apoptosis in disease pathogenesis and potentially identify new therapeutic targets.

What are the emerging research directions for TMEM214 functional characterization?

Several emerging research directions are expanding our understanding of TMEM214 function beyond its established role in ER stress-induced apoptosis:

• Post-translational modifications:

  • Investigation of whether TMEM214 undergoes phosphorylation, ubiquitination, or other modifications during ER stress

  • Identification of enzymes that regulate TMEM214 activity through post-translational modifications

  • Characterization of how these modifications affect TMEM214-procaspase 4 interactions

• Regulatory network mapping:

  • Identification of transcription factors that regulate TMEM214 expression

  • Investigation of microRNAs that may post-transcriptionally regulate TMEM214

  • Exploration of how TMEM214 expression is coordinated with other ER stress response factors

• Structure-function relationship studies:

  • Detailed structural analysis of the TMEM214-procaspase 4 binding interface

  • Investigation of conformational changes in TMEM214 during ER stress

  • Development of small molecule modulators of the TMEM214-procaspase 4 interaction

• Additional protein interactions:

  • Identification of other TMEM214-interacting proteins beyond procaspase 4

  • Investigation of whether TMEM214 participates in larger protein complexes at the ER membrane

  • Determination of whether TMEM214 has functions beyond apoptosis regulation

• Species-specific differences:

  • Comparative analysis of TMEM214 function across species

  • Evaluation of whether TMEM214 function in mice models accurately reflects human biology

  • Assessment of evolutionary conservation of the TMEM214-procaspase 4 interaction

These emerging research directions will provide a more comprehensive understanding of TMEM214 biology and may reveal novel therapeutic opportunities for diseases involving ER stress dysregulation.