TMEM173 Antibody, HRP conjugated

What is TMEM173/STING and why is it significant in research?

TMEM173, more commonly known as STING (Stimulator of Interferon Genes), is a transmembrane adaptor protein that plays a critical role in innate immune signaling. It functions as a sensor of cytosolic DNA from bacteria and viruses, promoting the production of type I interferons (IFN-alpha and IFN-beta). STING is widely expressed in various cell types including endothelial cells, epithelial cells, T cells, macrophages, and dendritic cells, with predominant localization in the endoplasmic reticulum (ER). Its significance in research stems from its central role in cytosolic DNA sensing pathways, making it a valuable target for studying innate immune responses to pathogens, autoimmune diseases, and cancer immunosurveillance .

What are the key differences between polyclonal and monoclonal TMEM173/STING antibodies?

Polyclonal TMEM173/STING antibodies, such as the HRP-conjugated rabbit polyclonal antibody (CSB-PA023754LB01HU), are derived from multiple B-cell lineages and recognize multiple epitopes on the STING protein. This provides robust signal detection across various applications and potentially greater tolerance to protein denaturation. In contrast, monoclonal antibodies like the mouse IgG2a monoclonal (clone O94E12) are produced from a single B-cell clone, recognizing a single epitope with high specificity. This makes monoclonal antibodies ideal for distinguishing between closely related proteins or specific conformational states of STING. For experimental approaches requiring consistent lot-to-lot reproducibility and high specificity, monoclonal antibodies are often preferred, while polyclonal antibodies may provide greater sensitivity, especially in applications where the protein may be partially denatured or in low abundance .

What cell lines and sample types are most appropriate for TMEM173/STING detection?

Based on validated research applications, several human cell lines have demonstrated reliable TMEM173/STING expression and are appropriate for antibody validation and experimental studies. THP-1 (human acute monocytic leukemia) and U937 (human histiocytic lymphoma) cell lines show consistent STING expression detectable by Western blot at approximately 40 kDa and by flow cytometry following appropriate fixation and permeabilization. HepG2 (human liver cancer) and HT-29 (human colorectal adenocarcinoma) cells have been successfully used for immunofluorescence applications with positive detection of STING. For advanced applications investigating STING's role in innate immunity, HCT116 cells have been utilized to study PARP1 depletion effects on STING activation and interferon response gene expression. Primary cell types including macrophages, dendritic cells, endothelial cells, and epithelial cells naturally express STING and can be appropriate for studies examining physiological functions. When studying STING in tissue contexts, proper fixation protocols (typically paraformaldehyde) and permeabilization (commonly with saponin) are critical for successful antibody penetration and target recognition .

How can I optimize intracellular staining for flow cytometry using TMEM173/STING antibodies?

Optimizing intracellular staining for TMEM173/STING detection by flow cytometry requires careful attention to fixation, permeabilization, and antibody incubation conditions. Begin with cell preparation by collecting approximately 1×10^6 cells per sample, washing in PBS containing 1% BSA, and fixing with 4% paraformaldehyde for 10-15 minutes at room temperature. For STING, which is predominantly localized in the endoplasmic reticulum, effective permeabilization is critical—use 0.1-0.5% saponin in PBS/BSA buffer for 10-15 minutes, as this has been validated for STING detection in THP-1 and U937 cell lines. When using HRP-conjugated antibodies, ensure complete permeabilization for optimal access to intracellular targets. Incubate with the primary antibody at the recommended concentration (approximately 0.40 μg per 10^6 cells for most STING antibodies) for 30-60 minutes at room temperature or at 4°C overnight if needed. If using unconjugated antibodies, follow with appropriate fluorochrome-conjugated secondary antibody incubation. Include proper controls—isotype control antibodies (such as MAB0041 when using mouse monoclonals) to establish background staining levels, and positive control cell lines with known STING expression. During analysis, use appropriate gating strategies to exclude dead cells and debris, and consider co-staining with ER markers to confirm STING localization patterns .

How do I select between different TMEM173/STING antibody clones for specific research applications?

Selecting the optimal TMEM173/STING antibody clone requires careful consideration of the specific research application, target epitope, and experimental conditions. For applications requiring detection of full-length STING protein, select antibodies raised against extensive protein fragments, such as those targeting Ala215-Ser379 regions, which have been validated in Western blot applications showing characteristic ~40 kDa bands in human cell lines. For domain-specific studies, consider epitope mapping information—some clones specifically recognize the C-terminal domain critical for downstream signaling interactions, while others target N-terminal regions. The clone 723505 (MAB7169), validated for Western blot, flow cytometry, and immunoprecipitation applications, demonstrates robust performance across multiple techniques and might be suitable for multi-modal experimental designs. For immunofluorescence applications, fluorophore-conjugated antibodies like CL488-19851 offer direct detection capabilities in fixed cells. Consider species cross-reactivity needs—while most antibodies are validated for human STING, specific clones show cross-reactivity with mouse samples, enabling translational research between human and murine models. Additionally, for co-immunoprecipitation studies investigating STING interaction partners, select antibodies validated for immunoprecipitation with minimal heavy chain interference in subsequent analyses .

What controls should be included when using TMEM173/STING antibodies in experiments?

Implementing comprehensive controls is essential for rigorous experimental design when using TMEM173/STING antibodies. Primary negative controls should include isotype-matched control antibodies (such as MAB0041 for mouse IgG or appropriate rabbit IgG controls) processed identically to experimental samples to account for non-specific binding. Positive controls should incorporate cell lines with confirmed STING expression, such as THP-1, U937, or HepG2 cells, which have been validated across multiple studies. For genetic validation, consider STING-knockout or knockdown models alongside wild-type samples—this approach is particularly valuable for antibody validation and specificity confirmation. When studying STING activation, include appropriate stimulation controls: cells treated with known STING agonists (e.g., cGAMP, c-di-GMP) compared to untreated cells can verify functional responses. For subcellular localization studies, co-staining with established organelle markers (particularly ER markers, given STING's predominant localization) provides critical contextual information. In quantitative applications like Western blotting or flow cytometry, include loading controls (housekeeping proteins) or viability markers respectively to normalize results and account for technical variations. Finally, absorption controls, where the antibody is pre-incubated with excess target antigen before sample application, can further demonstrate binding specificity .

How can I quantitatively assess TMEM173/STING expression using HRP-conjugated antibodies?

Quantitative assessment of TMEM173/STING expression using HRP-conjugated antibodies requires systematic approaches across different experimental platforms. For Western blot quantification, implement densitometric analysis of the characteristic ~40 kDa STING band relative to loading controls (β-actin, GAPDH) using software like ImageJ, with standard curves generated from recombinant STING protein standards if absolute quantification is required. For ELISA applications, particularly suited to HRP-conjugated antibodies like CSB-PA023754LB01HU, develop a sandwich ELISA with a capture antibody targeting a different STING epitope than the HRP-conjugated detection antibody, using serial dilutions of recombinant STING protein to establish a standard curve—typical detection ranges for optimized ELISA protocols span 0.1-1000 ng/mL with sigmoidal 4PL regression models for quantification. For flow cytometric quantification, convert mean fluorescence intensity values to molecules of equivalent soluble fluorochrome (MESF) using calibration beads, or implement staining index calculations to normalize signal across experiments. For immunohistochemical quantification, employ digital pathology approaches with algorithms that quantify DAB signal intensity and distribution in tissue sections. Across all methods, biological replicates (minimum n=3) and technical replicates are essential for statistical robustness, with data typically analyzed using appropriate statistical tests (t-test, ANOVA) depending on experimental design and data distribution characteristics .

Why might I observe inconsistent or unexpected results with TMEM173/STING detection?

Inconsistent or unexpected results with TMEM173/STING detection can stem from multiple sources requiring systematic troubleshooting. Protein degradation is a common issue—STING is sensitive to proteolytic degradation, so ensure samples contain complete protease inhibitor cocktails and are maintained at appropriate temperatures throughout processing. STING exhibits cell-type specific expression patterns that vary considerably across tissues and cellular activation states; baseline expression in some cell types may be below detection limits until stimulated with appropriate agonists. Post-translational modifications, particularly phosphorylation of STING following activation, can alter antibody binding efficacy—consider phospho-specific antibodies when studying activated STING. Insufficient permeabilization is particularly problematic for STING detection given its ER localization; optimize permeabilization protocols with saponin (0.1-0.5%) or Triton X-100 (0.1-0.2%) depending on the application. Cross-reactivity with structurally similar proteins can occur, particularly with polyclonal antibodies; validate specificity using STING-knockout controls. For HRP-conjugated antibodies specifically, enzymatic activity can be compromised by improper storage (avoid repeated freeze-thaw cycles) or exposure to inhibitors present in buffers. When analyzing discrepancies between methods (e.g., Western blot vs. immunofluorescence), remember that STING undergoes conformational changes and forms aggregates upon activation, potentially masking epitopes in certain contexts .

How can I distinguish between inactive and activated forms of TMEM173/STING?

Distinguishing between inactive and activated forms of TMEM173/STING requires targeted experimental approaches that detect the conformational, localization, and post-translational changes that occur during activation. Phosphorylation state analysis is fundamental—upon activation, STING becomes phosphorylated at several residues (particularly Ser366 in human STING) by TBK1; phospho-specific antibodies that selectively recognize these modifications can directly identify activated STING. Subcellular localization tracking is equally informative—inactive STING predominantly localizes to the ER, while activated STING translocates to perinuclear regions, specifically ER-Golgi intermediate compartments; using immunofluorescence with co-staining for organelle markers can visualize this translocation. Conformational changes can be detected through differential epitope exposure—some epitopes become masked or exposed during STING activation, so using multiple antibodies targeting different regions may reveal activation status. Downstream signaling assessment provides functional evidence—activated STING induces phosphorylation of IRF3 and expression of type I interferons, which can be measured by phospho-IRF3 antibodies and IFN-β ELISA respectively. For biochemical approaches, non-reducing vs. reducing gel conditions can distinguish between monomeric and dimeric/oligomeric STING forms, as activation promotes dimerization. Additionally, STING undergoes palmitoylation upon activation, which can be detected through metabolic labeling with palmitate analogs or acyl-biotin exchange assays .

What are the most common technical pitfalls when using HRP-conjugated TMEM173/STING antibodies in ELISA?

When using HRP-conjugated TMEM173/STING antibodies in ELISA, researchers commonly encounter several technical pitfalls that can compromise results. Suboptimal coating conditions represent a primary challenge—STING protein or anti-STING capture antibodies may not efficiently adhere to plates; optimize coating buffer pH (typically 9.6 for carbonate buffer) and temperature (4°C overnight often yields better results than shorter room temperature incubations). Hook effect occurs when high antigen concentrations paradoxically produce decreased signal; implement sample dilution series to identify optimal detection ranges. HRP enzyme inhibition can result from sample components or contaminated buffers containing sodium azide or certain reducing agents; ensure all buffers are compatible with enzymatic activity. Non-specific binding causes elevated background; optimize blocking conditions (typically 1-5% BSA or milk protein) and consider adding 0.05% Tween-20 to washing and diluent buffers. Signal saturation occurs when substrate conversion exceeds the linear range; optimize substrate incubation time and employ stopping solutions at appropriate timepoints. Antibody aggregation is particularly problematic with direct HRP-conjugates; centrifuge antibody solutions before use and maintain proper storage conditions. Lot-to-lot variability in conjugation efficiency impacts quantitative reproducibility; validate each new lot against standard curves. For sandwich ELISA configurations, epitope masking can occur if capture and detection antibodies compete for overlapping regions; select antibody pairs targeting distinct STING domains. Finally, matrix effects from complex biological samples may interfere with antibody-antigen interactions; implement sample dilution in assay buffer and consider sample pre-clearance procedures if necessary .

How can TMEM173/STING antibodies be used to investigate innate immune signaling pathways?

TMEM173/STING antibodies serve as powerful tools for dissecting the complex machinery of innate immune signaling networks. For stimulus-dependent activation studies, researchers can track STING conformational changes and translocation following exposure to cytosolic DNA, cyclic dinucleotides (CDNs), or pathogen challenge using immunofluorescence with antibodies targeting different STING domains. Signaling complex formation can be investigated through co-immunoprecipitation experiments where STING antibodies are used to pull down associated proteins like TBK1, IRF3, and cGAS, revealing dynamic interaction networks and their temporal regulation. For high-resolution mechanistic studies, proximity ligation assays using STING antibodies paired with antibodies against putative interaction partners provide quantitative spatial analysis of protein-protein interactions at subcellular resolution. Chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against IRF3 following STING activation reveals genome-wide transcriptional responses. For systems-level analysis, mass spectrometry of STING immunoprecipitates can identify novel interaction partners and post-translational modifications. In translational applications, tissue microarray analysis with STING antibodies across disease states can correlate expression patterns with pathological outcomes. Recent advanced approaches include integrating STING antibodies into multiplexed imaging platforms (e.g., Imaging Mass Cytometry, CODEX) to analyze STING pathway components within preserved tissue architecture contexts, revealing cell type-specific activation patterns in complex tissues .

What role does TMEM173/STING play in coagulation during sepsis, and how can antibodies help study this function?

TMEM173/STING plays a critical and previously underappreciated role in driving lethal coagulation during sepsis through mechanisms independent of its canonical type I interferon response. Recent research has revealed that myeloid TMEM173 regulates coagulation in bacterial infections through a distinct pathway: TMEM173 binding to ITPR1 controls calcium release from the endoplasmic reticulum in macrophages and monocytes, with the resulting increase in cytosolic calcium driving Gasdermin D (GSDMD) cleavage and activation. This activated GSDMD subsequently triggers the release of F3 (tissue factor), the key initiator of blood coagulation. TMEM173 antibodies serve as crucial tools for investigating this pathway through several methodological approaches. Co-immunoprecipitation studies using STING antibodies can capture and identify calcium regulatory protein complexes, particularly ITPR1 interactions. Calcium flux imaging combined with immunostaining can correlate STING activation with intracellular calcium dynamics at single-cell resolution. For mechanistic dissection, STING antibodies enable visualization of subcellular localization changes during septic conditions, particularly translocation events between ER and other compartments. Western blotting with STING and GSDMD antibodies can track the activation sequence from STING stimulation to GSDMD cleavage. Flow cytometry with multiple markers can characterize cell populations contributing to coagulation in animal models of sepsis. Importantly, genetic or pharmacological inhibition of the TMEM173-GSDMD-F3 pathway has been shown to block systemic coagulation and improve animal survival in multiple sepsis models, indicating potential therapeutic applications that could be monitored using these antibody-based techniques .

How can emerging technologies enhance the application of TMEM173/STING antibodies in research?

Emerging technologies are dramatically expanding the capabilities and applications of TMEM173/STING antibodies in cutting-edge research. Single-cell proteomics approaches, particularly mass cytometry (CyTOF) incorporating metal-conjugated STING antibodies, enable high-dimensional analysis of STING pathway components across heterogeneous cell populations with simultaneous measurement of up to 40 proteins, revealing cell-specific activation signatures impossible to detect in bulk analyses. Super-resolution microscopy techniques (STORM, PALM, STED) combined with fluorophore-conjugated STING antibodies now visualize nanoscale organization of STING within the ER membrane and during translocation events, with resolution below 50nm revealing previously undetectable structural arrangements. Spatial transcriptomics integrated with STING immunohistochemistry correlates protein localization with gene expression profiles in tissue contexts, creating comprehensive spatial-molecular maps of STING-dependent responses. Bioorthogonal chemistry approaches enable pulse-chase labeling of newly synthesized STING protein through incorporation of modified amino acids, followed by click chemistry and antibody detection to track protein turnover rates during activation. Microfluidic platforms incorporating STING antibody-based detection enable real-time monitoring of single-cell activation dynamics with minimal sample input. For translational applications, extracellular vesicle (EV) analysis using anti-STING antibodies has revealed STING pathway components in circulating EVs that may serve as biomarkers. Machine learning algorithms applied to multiplexed imaging data from STING antibody panels can identify subtle phenotypic signatures predictive of disease outcomes. Additionally, CRISPR-based screening coupled with high-content imaging using STING antibodies enables genome-wide identification of regulators impacting STING localization and activation .

Comparison of TMEM173/STING Antibody Applications and Performance Characteristics

This table summarizes key TMEM173/STING antibodies discussed in the search results, comparing their characteristics and optimal applications for research use .

TMEM173/STING Molecular Characteristics and Expression Profile

This table provides essential information about the TMEM173/STING protein, including molecular characteristics, cellular expression patterns, and functional properties relevant to antibody-based detection and analysis .

Troubleshooting Guide for TMEM173/STING Antibody Applications

This comprehensive troubleshooting guide addresses common issues encountered when using TMEM173/STING antibodies across different experimental applications, with specific recommendations for resolving technical challenges .