TMEM173 Antibody, FITC conjugated

What is TMEM173/STING and why is it important to study?

TMEM173/STING is a 40-42 kDa four-transmembrane protein that functions as a critical mediator of both antiviral and MHC-II antigen recognition responses. It is predominantly located in the endoplasmic reticulum where it serves as an adaptor protein for intracellular viral detection molecules, participating in the induction of type I interferon responses . STING also plays a potential role in initiating apoptosis following MHC-II engagement. Human STING is 379 amino acids in length, containing an N-terminal cytoplasmic region (aa 1-20), four transmembrane segments (aa 21-173), and a C-terminal cytoplasmic domain (aa 174-379) . Studying STING is essential for understanding innate immune signaling pathways, particularly those involved in cytosolic DNA sensing and antiviral responses.

Which cell types express TMEM173/STING protein?

TMEM173/STING is expressed in various immune cells, including B cells, dendritic cells, macrophages, and monocytes . In experimental settings, STING expression has been confirmed in several cell lines, including THP-1 human acute monocytic leukemia cells, U937 human histiocytic lymphoma cells , HT-29 cells, and HepG2 cells . This distribution reflects STING's important role in innate immune surveillance across multiple tissue types and immune cell populations.

What are the typical applications for FITC-conjugated TMEM173 antibodies?

FITC-conjugated TMEM173 antibodies are versatile tools with several key applications:

These applications enable researchers to visualize STING localization, quantify expression levels, and study its dynamics in various experimental contexts.

What are the optimal sample preparation protocols for FITC-conjugated TMEM173 antibodies?

For intracellular staining applications like flow cytometry or immunofluorescence, proper fixation and permeabilization are crucial for detecting TMEM173/STING. The most effective protocol involves:

• Cell fixation with paraformaldehyde (typically 2-4%) to preserve cellular architecture

• Permeabilization with saponin (0.1-0.5%) to allow antibody access to intracellular compartments

• Blocking with appropriate serum (5-10%) to reduce non-specific binding

• Incubation with FITC-conjugated TMEM173 antibody at recommended dilutions (typically 1:50-1:500 for IF/ICC)

• Thorough washing to remove unbound antibody

This methodology has been validated for detecting STING in multiple cell types, including human peripheral blood mononuclear cell (PBMC) monocytes and THP-1 cells .

How should FITC-conjugated TMEM173 antibodies be stored to maintain optimal performance?

To maintain the integrity and performance of FITC-conjugated TMEM173 antibodies, the following storage conditions are recommended:

• Aliquot the antibody upon first use to minimize freeze-thaw cycles

• Store at -20°C in the dark to prevent photobleaching of the FITC fluorophore

• Include cryoprotectants such as glycerol (typically 50%) in the storage buffer

• Avoid repeated freeze/thaw cycles which can damage both the antibody and the fluorophore

• When working with the antibody, keep it on ice and protected from light

Following these guidelines will help maintain antibody activity and fluorescence intensity over time, ensuring consistent experimental results.

What controls should be included when using FITC-conjugated TMEM173 antibodies?

Rigorous experimental design requires appropriate controls when using FITC-conjugated TMEM173 antibodies:

• Isotype control: Use an isotype-matched antibody (e.g., rabbit IgG for polyclonal rabbit antibodies) conjugated to FITC at the same concentration to assess non-specific binding

• Negative control cells: Include cells known to express minimal/no TMEM173 protein

• Positive control cells: Include validated cell lines known to express TMEM173 (e.g., THP-1, U937, HepG2)

• Blocking peptide control: Pre-incubate the antibody with its immunogen peptide to confirm specificity

• Knockdown/knockout validation: Where possible, use TMEM173 knockdown or knockout cells to verify specificity

These controls help distinguish specific signal from background and validate antibody performance across different experimental conditions.

How can FITC-conjugated TMEM173 antibodies be optimized for multi-parameter flow cytometry?

For multi-parameter flow cytometry experiments involving FITC-conjugated TMEM173 antibodies:

• Consider fluorophore compensation: FITC's emission spectrum (peak at 515 nm) may overlap with other commonly used fluorophores like PE. Proper compensation controls are essential to correct for spillover

• Optimize antibody concentration: Titrate the FITC-conjugated TMEM173 antibody (typically starting at 0.25 μg per 10^6 cells) to determine the optimal signal-to-noise ratio

• Adjust fixation and permeabilization conditions: Different fixation/permeabilization reagents may be necessary depending on the markers being co-stained

• Select complementary fluorophores: Pair FITC (excited by 488 nm laser) with fluorophores excited by different lasers (e.g., APC, PE-Cy7) to minimize compensation requirements

• Include FMO (Fluorescence Minus One) controls to set accurate gating boundaries

This approach has been validated for analyzing STING expression in human PBMC monocytes and various cell lines .

What strategies can address the challenge of detecting low abundance TMEM173/STING protein?

For detecting low levels of TMEM173/STING expression:

• Signal amplification: Consider using secondary detection systems with multiple fluorophores per secondary antibody

• Extended incubation times: Increase primary antibody incubation time (e.g., overnight at 4°C) to enhance binding to low abundance targets

• Concentration optimization: Use higher antibody concentrations while monitoring background signals

• Cell stimulation: Pre-treat cells with stimuli known to upregulate STING expression, such as cGAMP or poly(dA:dT)

• Enhanced imaging techniques: For microscopy applications, use techniques like confocal microscopy with increased exposure times or signal averaging

Combining these approaches can significantly improve detection sensitivity while maintaining specificity for TMEM173/STING protein.

How can FITC-conjugated TMEM173 antibodies be used to study STING trafficking in response to stimulation?

To investigate STING trafficking dynamics:

• Time-course experiments: Stimulate cells with cGAMP or other STING agonists and fix cells at various time points (0-24 hours)

• Co-localization analysis: Combine FITC-conjugated TMEM173 antibody with markers for different cellular compartments (ER, Golgi, endosomes, etc.)

• Live-cell imaging: For cell lines, consider membrane-permeable STING antibody fragments for real-time tracking

• Subcellular fractionation: Complement imaging with biochemical fractionation followed by Western blotting

• Quantitative image analysis: Use software to quantify changes in STING distribution patterns before and after stimulation

These approaches enable detailed analysis of how STING relocates from the ER to other compartments during immune signaling.

How can background fluorescence be minimized when using FITC-conjugated TMEM173 antibodies?

To reduce background fluorescence in experiments with FITC-conjugated TMEM173 antibodies:

• Optimize blocking: Use 5-10% serum from the species of the secondary antibody (if used) or BSA to block non-specific binding sites

• Autofluorescence quenching: Treat samples with 0.1-1% sodium borohydride or commercial autofluorescence quenchers before antibody incubation

• Washing optimization: Increase the number and duration of washes with detergent-containing buffer (e.g., 0.05-0.1% Tween-20)

• Antibody titration: Determine the minimum antibody concentration that gives specific signal to reduce non-specific binding

• Fixative selection: Consider different fixatives, as some can introduce autofluorescence (aldehydes are particularly problematic)

These strategies help ensure that the observed fluorescence signal is specific to TMEM173/STING rather than technical artifacts.

What are the key considerations for validating FITC-conjugated TMEM173 antibody specificity?

Rigorous validation of FITC-conjugated TMEM173 antibody specificity should include:

• Western blot confirmation: Verify that the antibody detects a band of the expected size (40-42 kDa for monomeric STING; approximately 80 kDa for dimeric STING)

• Knockout/knockdown controls: Test the antibody on STING-deficient cells to confirm absence of signal

• Peptide competition: Pre-incubate the antibody with excess immunizing peptide to demonstrate signal ablation

• Cross-reactivity testing: Test on cells from different species if cross-reactivity is claimed

• Comparison with alternative antibody clones: Confirm staining pattern using antibodies targeting different epitopes of STING

This comprehensive validation approach ensures confidence in experimental results and minimizes the risk of misinterpreting non-specific signals.

What factors affect the variability in FITC-conjugated TMEM173 antibody performance across experiments?

Several factors can contribute to variability in FITC-conjugated TMEM173 antibody performance:

• Photobleaching: FITC is relatively prone to photobleaching. Minimize exposure to light during all steps and use anti-fade mounting media for microscopy

• Storage conditions: Improper storage (multiple freeze-thaw cycles, exposure to light) can diminish antibody performance

• Buffer composition: pH and salt concentration can affect antibody binding and FITC fluorescence intensity

• Fixation effects: Different fixation methods and durations can alter epitope accessibility and fluorescence properties

• Lot-to-lot variability: Different antibody lots may show slight variations in performance and optimal concentrations

Maintaining consistent experimental conditions and including appropriate controls with each experiment can help mitigate these sources of variability.

How can FITC-conjugated TMEM173 antibodies be integrated into high-content screening approaches?

FITC-conjugated TMEM173 antibodies can enhance high-content screening workflows through:

• Automated imaging platforms: Utilize high-throughput microscopy systems to quantify STING expression and localization across multiple treatment conditions

• Multiparametric analysis: Combine STING detection with other cellular markers to create multidimensional phenotypic profiles

• Machine learning classification: Train algorithms to identify subtle changes in STING distribution patterns

• Dose-response studies: Systematically evaluate compound effects on STING activation across concentration ranges

• Time-lapse experiments: Track dynamic changes in STING localization following stimulation in fixed timepoint series

These approaches enable comprehensive analysis of how diverse stimuli and experimental conditions affect STING biology in various cell types.

What experimental approaches can elucidate the relationship between STING localization and functional activation?

To study the relationship between STING localization and activation:

• Co-localization with phospho-specific markers: Use antibodies against phosphorylated STING (pSer358) alongside FITC-conjugated total STING antibodies

• Functional readouts: Correlate STING localization with downstream functional outcomes, such as IRF3 nuclear translocation or type I interferon production

• Structure-function studies: Compare localization patterns of wild-type STING versus mutant variants with altered functionality

• Super-resolution microscopy: Apply techniques like STORM or STED to resolve nanoscale changes in STING distribution not visible by conventional microscopy

• Proximity ligation assays: Detect interactions between STING and binding partners in different subcellular compartments

These approaches provide mechanistic insights into how STING localization correlates with its activation status and downstream signaling capacity.