TMEM173 Antibody

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

TMEM173 (Transmembrane Protein 173), commonly known as STING (Stimulator of Interferon Genes), is a critical adaptor protein involved in innate immune responses. This protein functions as a sensor for cytosolic DNA and plays a crucial role in type I interferon production pathways. STING is encoded by the TMEM173 gene and may also be referred to as NET23, MPYS, MITA, or ERIS in literature .

The significance of STING lies in its central role in immune surveillance and host defense against pathogens, particularly in detecting cytosolic DNA. Research on STING has expanded significantly due to its implications in autoimmune diseases, cancer immunotherapy, and inflammatory conditions .

What are the typical molecular characteristics of human TMEM173/STING protein?

Human TMEM173/STING protein has the following molecular characteristics:

• Molecular weight: Approximately 37-42 kDa (observed in Western blot analysis)

• Amino acid length: 379 amino acids

• Gene ID (NCBI): 340061

• UniProt ID: Q86WV6

• Structure: Transmembrane protein with both cytosolic and membrane-spanning domains

• Accession number: BC047779

In some experimental conditions, particularly in Western blot applications, STING may also appear as a band at approximately 80 kDa, which may represent dimerized or post-translationally modified forms of the protein .

What cell types and tissues commonly express TMEM173/STING?

TMEM173/STING expression has been documented in multiple cell types and tissues:

Data compiled from multiple sources .

What criteria should researchers consider when selecting a TMEM173/STING antibody for their experiments?

When selecting a TMEM173/STING antibody, researchers should consider several critical factors:

• Application compatibility: Ensure the antibody has been validated for your specific application (WB, IHC, IF, FC, IP)

• Species reactivity: Verify cross-reactivity with your experimental model (human, mouse, rat)

• Epitope recognition: Consider which domain of STING the antibody recognizes (e.g., Ala215-Ser379)

• Clone type: Determine whether monoclonal (greater specificity) or polyclonal (broader epitope recognition) is appropriate for your research question

• Validation methods: Check for knockout (KO) validation, which provides strong evidence of specificity

• Citation record: Review publications that have used the antibody in similar applications

• Format and conjugation: Select appropriate formats (unconjugated vs. conjugated with fluorophores)

A critical step in antibody selection is reviewing validation data, including Western blot images showing the expected molecular weight band (37-42 kDa for STING) .

How can researchers validate the specificity of a TMEM173/STING antibody?

Proper validation of TMEM173/STING antibody specificity involves multiple complementary approaches:

• Knockout controls: Use STING knockout cell lines as negative controls (e.g., STING/TMEM173 knockout HeLa cell line)

• Knockdown verification: Compare antibody signal in cells treated with STING-specific siRNA/shRNA versus control

• Multiple antibody comparison: Use antibodies targeting different epitopes of STING and compare detection patterns

• Expected molecular weight verification: Confirm detection at the expected molecular weight (37-42 kDa)

• Immunoprecipitation validation: Perform IP followed by Western blot with a different STING antibody

• Peptide competition assay: Pre-incubate the antibody with a blocking peptide to confirm specificity

• Cross-reactivity testing: Test antibody against related proteins to ensure it doesn't cross-react

Example validation data: Simple Western analysis showing specific STING/TMEM173 detection at approximately 41 kDa in parental HeLa cells but not in STING/TMEM173 knockout HeLa cells .

What are the optimal conditions for Western blot detection of TMEM173/STING?

Optimal Western blot conditions for TMEM173/STING detection:

Critical consideration: STING protein expression can be highly variable between cell types; THP-1, U937, and PBMC monocytes typically show strong expression .

What are the recommended protocols for immunohistochemical detection of TMEM173/STING in tissue samples?

Recommended protocol for TMEM173/STING immunohistochemistry:

• Tissue preparation:

  • Fixation: 10% neutral buffered formalin (18-24 hours)

  • Processing: Standard paraffin embedding

  • Sectioning: 4-5 μm thick sections

• Antigen retrieval (critical for STING detection):

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heating method: Pressure cooker (20 minutes) or microwave (15 minutes)

• Blocking and antibody incubation:

  • Peroxidase blocking: 3% hydrogen peroxide (10 minutes)

  • Protein blocking: 5-10% normal serum from secondary antibody species (1 hour)

  • Primary antibody dilution: 1:500-1:8000 (antibody dependent)

  • Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Detection system:

  • HRP-polymer detection system

  • DAB chromogen (5-10 minutes) for brightfield visualization

  • Counterstain with hematoxylin (light staining to maintain contrast)

• Controls:

  • Positive tissue control: Human tonsillitis tissue, human spleen tissue (known to express STING)

  • Negative control: Primary antibody omission or isotype control

Specialized consideration: For multiplex immunofluorescence detection of STING in human brain cortex, the COMET™ platform with Alexa Fluor™ Plus 647 secondary antibody has been validated with DAPI counterstain .

What are the key methodological considerations for flow cytometric analysis of TMEM173/STING?

Flow cytometric detection of TMEM173/STING requires special attention to permeabilization since STING is primarily an intracellular protein:

• Cell preparation:

  • Single cell suspension: Mechanical dissociation for tissues; harvesting for adherent cells

  • Fixation: 4% paraformaldehyde (15-20 minutes at room temperature)

  • Critical step: Permeabilization with saponin (0.1-0.5%) to access intracellular STING

• Staining protocol:

  • Blocking: 5% normal serum in permeabilization buffer (30 minutes)

  • Primary antibody: 0.2-0.4 μg per 10^6 cells

  • Incubation: 30-60 minutes at room temperature

  • Secondary detection: Allophycocyanin-conjugated secondary antibody

• Controls and analysis:

  • Isotype control: Crucial for setting negative population gates (e.g., MAB0041 can be used as isotype control)

  • Positive control: THP-1 or U937 cells (known to express STING)

  • Analysis gates: Sequential gating strategy to identify single, viable cells before STING assessment

Example: "Detection of STING/TMEM173 in Human PBMC Monocytes by Flow Cytometry. Human peripheral blood mononuclear cell (PBMC) monocytes were stained with Mouse Anti-Human STING/TMEM173 Monoclonal Antibody (Catalog # MAB7169, filled histogram) or isotype control antibody (MAB0041, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (F0101B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin."

How can TMEM173/STING antibodies be utilized to study STING pathway activation in disease models?

TMEM173/STING antibodies serve as valuable tools for investigating STING pathway activation in various disease models:

• Assessing phosphorylation-dependent activation:

  • Use antibodies that detect STING alongside phosphorylated TBK1 (pTBK1) and phosphorylated IRF3 (pIRF3) to monitor the complete signaling cascade

  • Example application: "Immunoblot analysis of STING, phosphorylated TBK1 (pTBK1), and pIRF3 in the cortex of mice without stroke or at day 3 after stroke."

• Cell-specific STING activation detection:

  • Multiplex immunofluorescence with cell-type markers and STING antibodies

  • Flow cytometric analysis of STING expression in specific immune cell populations

  • Applications include cancer immunology, neuroinflammation, and autoimmune disease models

• Therapeutic intervention assessment:

  • Monitor STING pathway modulation after treatment with STING agonists or antagonists

  • Example: "Immunoblot analysis of STING, pTBK1, and pIRF3 in isolated neutrophils for each treatment group (n = 5)."

• STING-associated vasculopathy studies:

  • Investigating STING expression in SAVI (STING-associated vasculopathy with onset in infancy)

  • Correlating STING activation with vascular damage markers

  • Example: "Confocal images of CD31-positive microvessels and quantification of microvascular density in the peri-infarct cortex at 14 days in mice treated with control IgG or IFNAR-neutralizing antibody, and STING shRNA or control adenovirus."

Advanced experimental design may include temporal analysis of STING activation following stimuli and correlation with downstream effects like inflammatory cytokine production.

What techniques can be employed to study STING protein-protein interactions using TMEM173 antibodies?

Advanced techniques for studying STING protein-protein interactions include:

• Co-immunoprecipitation (Co-IP):

  • Precipitate STING using specific antibodies (e.g., MAB7169, AF6516)

  • Western blot for interacting partners

  • Example protocol: "Immunoprecipitation was performed using 2.0 μg of Mouse Anti-Human STING/TMEM173 Monoclonal Antibody (Catalog # MAB7169) pre-coupled to Dynabeads protein G. Immunoprecipitated STING/TMEM173 was detected with a Rabbit Anti-STING/TMEM173 antibody."

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in situ using STING antibodies paired with antibodies against potential interacting partners

  • Visualization of interactions as fluorescent dots under microscopy

  • Particularly useful for studying STING interactions with cGAS, TBK1, or other signaling components

• Immunofluorescence co-localization:

  • Double immunofluorescence staining with STING antibodies and markers for subcellular compartments or interacting proteins

  • Example: Tracking STING translocation from ER to Golgi apparatus upon activation

  • Confocal microscopy analysis with correlation coefficients to quantify co-localization

• FRET/BRET-based interaction studies:

  • Engineering fluorescent protein-tagged STING constructs

  • Validating interactions using antibodies against endogenous proteins

  • Correlating FRET signals with functional outcomes of pathway activation

• Crosslinking mass spectrometry:

  • Chemical crosslinking of protein complexes

  • Immunoprecipitation with STING antibodies

  • Mass spectrometry identification of interacting partners

  • Validation of novel interactions with reciprocal Co-IP

These methodologies can reveal dynamic changes in STING's interactome during immune activation and disease states.

How can researchers troubleshoot inconsistent results with TMEM173/STING antibodies across different applications?

Troubleshooting strategies for inconsistent TMEM173/STING antibody results:

• Application-specific optimization:

  Application	Common Issue	Troubleshooting Approach
  Western Blot	Multiple bands	Optimize sample preparation; use reducing agents; try gradient gels
  IHC	Weak or no signal	Test different antigen retrieval methods (crucial: TE buffer pH 9.0)
  Flow Cytometry	Poor discrimination	Increase permeabilization with saponin; optimize fixation time
  IF/ICC	High background	Increase blocking time; test different blocking agents; optimize antibody concentration

• Antibody-specific considerations:

  • Different antibodies recognize different epitopes; switch to an antibody targeting a different domain if one fails

  • Epitope masking: Post-translational modifications may block antibody binding sites

  • Clone performance variability: Monoclonal antibody 66680-1-Ig shows superior performance in WB (1:5000-1:50000) compared to some other antibodies

• Sample-dependent factors:

  • STING expression levels vary dramatically between cell types

  • Activation state affects antibody accessibility (particularly for phospho-specific antibodies)

  • Fixation-sensitive epitopes: Some epitopes may be destroyed by overfixation

• Technical validation approaches:

  • Parallel testing with multiple antibodies on the same samples

  • Include known positive controls (e.g., THP-1 cells for human STING)

  • Use STING knockout samples as negative controls

  • Sequential optimization: Systematically vary one parameter at a time

Example case study: "Detection of Human STING/TMEM173 by Simple Western showed lysates of HeLa human cervical epithelial carcinoma parental cell line and STING/TMEM173 knockout HeLa cell line (KO), loaded at 0.2 mg/mL. A specific band was detected for STING/TMEM173 at approximately 41 kDa in the parental HeLa cell line, but is not detectable in knockout HeLa cell line." - This demonstrates the importance of proper controls for troubleshooting.

How are TMEM173/STING antibodies being utilized in the study of innate immune responses to cytosolic DNA?

TMEM173/STING antibodies are critical tools in studying innate immune responses to cytosolic DNA through several advanced applications:

• Kinetic analysis of STING pathway activation:

  • Serial immunoblotting with phospho-specific antibodies to track STING-TBK1-IRF3 activation cascade

  • Quantitative assessment of STING translocation following DNA stimulation

  • Example: Monitoring STING, pTBK1, and pIRF3 levels in ischemic cortex at different time points after stroke

• Cell type-specific STING responses:

  • Flow cytometric analysis of STING expression and activation in defined immune cell subsets

  • Single-cell analysis correlating STING levels with functional outcomes

  • Multi-parameter flow cytometry combining STING antibodies with cytokine detection

• STING-dependent NET (Neutrophil Extracellular Trap) formation:

  • Studying STING's role in neutrophil activation and NET formation

  • Combined immunofluorescence detection of STING and NET markers

  • As noted in research: "STING-mediated effects on vascular remodeling are due to NETs."

• Stimulus-specific STING activation patterns:

  • Comparing STING activation profiles following exposure to different DNA ligands

  • Differential detection of STING conformational changes upon binding various cyclic dinucleotides

  • Correlation with downstream interferon production

These approaches have advanced our understanding of STING's central role in innate immunity against pathogens and in sterile inflammation conditions.

What are the methodological considerations for using TMEM173/STING antibodies in multiplexed imaging techniques?

Methodological considerations for TMEM173/STING antibodies in multiplexed imaging:

• Antibody selection for multiplexing:

  • Species compatibility: Choose primary antibodies from different host species to avoid cross-reactivity

  • Isotype diversity: When using multiple antibodies from the same species, select different isotypes

  • Validated combinations: Test antibody panels on control samples before experimental use

• Sequential immunofluorescence (seqIF™) protocol optimization:

  • Example protocol: "STING/TMEM173 was detected in immersion fixed paraffin-embedded sections of human brain Cortex (Cerebrum) using Rabbit Anti-Human STING/TMEM173, Monoclonal Antibody (Catalog #NBP3-18816) at 10ug/mL at 37°C for 4 minutes. Before incubation with the primary antibody, tissue underwent an all-in-one dewaxing and antigen retrieval preprocessing using PreTreatment Module (PT Module) and Dewax and HIER Buffer H (pH 9; Epredia Catalog # TA-999-DHBH)."

  • Careful titration of each antibody in the multiplex panel is essential

  • Incorporate specificity controls for each marker in the panel

• Signal amplification and spectral separation:

  • Tyramide signal amplification (TSA) for low-abundance targets

  • Spectral unmixing for closely overlapping fluorophores

  • Selection of fluorophores with minimal spectral overlap (e.g., Alexa Fluor™ Plus 647 as used for STING detection )

• Specialized platforms for high-dimensional imaging:

  • COMET™ platform for automated sequential staining

  • Imaging mass cytometry (IMC) for highly multiplexed tissue analysis

  • CODEX system for co-detection of >40 proteins on a single tissue section

• Quantitative analysis approaches:

  • Single-cell segmentation for quantifying STING expression per cell

  • Spatial relationship analysis between STING and other markers

  • Machine learning-based classification of cell phenotypes based on marker patterns

These advanced approaches enable researchers to place STING activation in the context of complex cellular microenvironments and signaling networks.

What considerations should be made when studying post-translational modifications of TMEM173/STING using antibodies?

Studying post-translational modifications (PTMs) of TMEM173/STING requires specialized approaches:

• Phosphorylation-specific detection:

  • Use phospho-specific antibodies targeting known STING phosphorylation sites

  • Parallel detection of STING and its phosphorylated form

  • Example approach: "Immunoblot analysis of STING, phosphorylated TBK1 (pTBK1), and pIRF3 in the ischemic cortex at day 3."

  • Critical control: Lambda phosphatase treatment to confirm phospho-specificity

• Ubiquitination analysis:

  • Immunoprecipitation of STING followed by ubiquitin detection

  • Denaturing conditions required to disrupt non-covalent interactions

  • Pre-treatment with deubiquitinase inhibitors to preserve modifications

  • Example protocol adaptation: "Immunoprecipitation was performed using 2.0 μg of Mouse Anti-Human STING/TMEM173 Monoclonal Antibody pre-coupled to Dynabeads protein G" with additional deubiquitinase inhibitors

• Palmitoylation assessment:

  • STING palmitoylation affects its trafficking and activation

  • Acyl-biotin exchange (ABE) assay followed by STING immunoprecipitation

  • Hydroxylamine-sensitive detection indicates palmitoylation

• Sample preparation considerations:

  • Rapid sample collection and processing to preserve labile PTMs

  • Phosphatase inhibitors crucial for phosphorylation studies

  • Proteasome inhibitors for ubiquitination studies

  • N-ethylmaleimide (NEM) to preserve cysteine-dependent modifications

• Advanced mass spectrometry approaches:

  • Immunoprecipitation of STING using validated antibodies (e.g., AF6516, MAB7169)

  • LC-MS/MS analysis to identify multiple PTMs simultaneously

  • SILAC or TMT labeling for quantitative comparison of PTM changes upon stimulation

These methodologies enable researchers to understand the complex regulation of STING through dynamic post-translational modifications in different cellular contexts and disease states.