TMEM173 Antibody, Biotin conjugated

What is TMEM173 and why is it important in immunology?

TMEM173/STING is a 40-42 kDa 4-transmembrane protein that functions as a facilitator of innate immune signaling, promoting the production of type I interferons (IFN-alpha and IFN-beta). It plays a critical role in innate immune responses triggered by non-CpG double-stranded DNA from viruses and bacteria delivered to the cytoplasm. STING activates both NF-kappa-B and IRF3 transcription pathways to induce expression of type I interferons, thereby establishing an anti-viral state . Additionally, it may be involved in translocon function, potentially influencing type I interferon induction, and mediates death signaling via activation of the extracellular signal-regulated kinase (ERK) pathway . The significance of TMEM173 extends to pathological conditions, as mutations in the TMEM173 gene have been associated with STING-associated vasculopathy with onset in infancy (SAVI), an autoinflammatory disease characterized by early-onset inflammation leading to severe skin lesions .

What are the common applications for TMEM173 Antibody, Biotin conjugated?

TMEM173 Antibody, Biotin conjugated is utilized in multiple research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): Typically used at dilutions of 1:500-1000 for detecting TMEM173 in various sample types .

• Immunohistochemistry (IHC):

  • Paraffin-embedded tissues (IHC-P): Used at dilutions of 1:200-400

  • Frozen sections (IHC-F): Used at dilutions of 1:100-500

• Flow Cytometry: For intracellular staining of TMEM173 in various cell types, including human peripheral blood mononuclear cell (PBMC) monocytes, THP-1, and U937 cell lines, following fixation with paraformaldehyde and permeabilization with saponin .

• Western Blot: For detection of TMEM173 at approximately 37-40 kDa in human cell lysates, particularly from immune cell lineages like THP-1 and U937 .

• Immunoprecipitation: Effective at approximately 2 μg/mL when targeting TMEM173 in cell lysates such as PMA-treated THP-1 cells .

What is the subcellular localization of TMEM173 and how does this affect antibody selection?

TMEM173/STING exhibits a complex subcellular distribution pattern, primarily localized to the cytoplasm and cell membrane . It has been reported to associate with the endoplasmic reticulum and potentially mitochondria, as suggested by some of its alternative names: Endoplasmic reticulum interferon stimulator (ERIS) and Mitochondrial mediator of IRF3 activation . This subcellular distribution is critical for its function in innate immune signaling. When selecting antibodies for immunostaining applications, researchers must consider the need for membrane permeabilization protocols to access intracellular epitopes. For applications targeting TMEM173, fixation with paraformaldehyde followed by permeabilization with saponin has been successfully employed in flow cytometry studies . When designing experiments, the accessibility of the epitope in its native conformation must be considered, particularly for transmembrane proteins like TMEM173.

How do genetic variants of TMEM173 impact experimental design?

The human TMEM173 gene displays significant heterogeneity and population stratification that must be considered when designing experiments. The R232 variant is the most common human TMEM173 allele, but more than 50% of Americans are not R232/R232 homozygous . The second most common allele is HAQ (R71H-G230A-R293Q), and while R232/R232 is the dominant genotype in Europeans, R232/HAQ is most common in East Asians .

Research has shown that homozygous HAQ individuals account for approximately 16.1% of East Asians and 2.8% of Europeans, while being virtually absent in African populations . Importantly, HAQ/HAQ carriers express extremely low MPYS/STING protein levels and show decreased TMEM173 transcript expression, resulting in minimal response to cyclic dinucleotides . When designing experiments involving TMEM173 detection or functional studies, researchers must consider:

• The genetic background of cell lines or primary cells used

• Potential differences in antibody binding efficiency to different TMEM173 variants

• Variations in protein expression levels across different genetic backgrounds

• The need for genotyping samples when studying functional responses dependent on TMEM173

How should I optimize Western blot protocols for TMEM173 Antibody, Biotin conjugated?

Optimizing Western blot protocols for TMEM173 Antibody, Biotin conjugated requires careful consideration of several parameters:

• Sample Preparation:

  • Use appropriate lysis buffers compatible with membrane proteins (Immunoblot Buffer Group 1 has been successfully used)

  • Include protease inhibitors to prevent degradation of TMEM173

  • Perform lysis under reducing conditions for optimal epitope exposure

• Gel Electrophoresis and Transfer:

  • Use PVDF membranes for optimal protein binding and signal strength

  • TMEM173 is detected at approximately 37-40 kDa; ensure appropriate molecular weight markers

  • Consider gradient gels (10-12%) for better resolution in this molecular weight range

• Antibody Concentration and Detection:

  • Optimal primary antibody concentration: 0.2 μg/mL as demonstrated in successful experiments

  • Secondary detection: Use HRP-conjugated anti-species antibodies (such as Anti-Mouse IgG for mouse monoclonal antibodies)

  • For biotin-conjugated antibodies, use streptavidin-HRP for detection

  • Include positive controls like THP-1 or U937 human cell lysates, which show good TMEM173 expression

• Signal Development:

  • Use enhanced chemiluminescence (ECL) for sensitive detection

  • Optimize exposure times based on signal strength to avoid saturation

To validate specificity, consider running parallel blots with isotype controls and samples known to have differential TMEM173 expression levels.

What are the recommended fixation and permeabilization protocols for intracellular TMEM173 staining?

For optimal intracellular TMEM173 staining, especially in flow cytometry applications, the following protocol has been validated:

• Cell Preparation:

  • For suspension cells (e.g., THP-1, U937, PBMCs): Collect cells at concentration of 1-10 × 10^6 cells/mL

  • For adherent cells: Detach using enzyme-free dissociation buffers to preserve surface epitopes

• Fixation:

  • Paraformaldehyde (PFA) fixation: Use 4% PFA for 10-15 minutes at room temperature

  • This preserves cellular structure while enabling antibody access to intracellular compartments

• Permeabilization:

  • Saponin has been successfully used for TMEM173 detection

  • Typically used at 0.1-0.5% concentration in PBS/BSA buffer

  • Maintain saponin in all subsequent wash and antibody incubation steps as its effect is reversible

• Antibody Staining:

  • For flow cytometry: Use 0.25 μg antibody per 10^6 cells

  • Include appropriate isotype controls (e.g., MAB0041 has been used as control for MAB7169)

  • Incubate 30-60 minutes at room temperature or 4°C

  • For biotin-conjugated antibodies, follow with fluorophore-conjugated streptavidin

• Detection:

  • For biotin-conjugated antibodies in flow cytometry, secondary detection with streptavidin conjugated to appropriate fluorophores

  • For immunocytochemistry, similar fixation/permeabilization protocols apply, with recommended antibody concentrations of 8-25 μg/mL

This protocol has been validated on multiple cell types including human peripheral blood mononuclear cell (PBMC) monocytes, THP-1 human acute monocytic leukemia cell line, and U937 human histiocytic lymphoma cell line .

How can I validate the specificity of TMEM173 Antibody, Biotin conjugated?

Validating antibody specificity is critical for reliable research results. For TMEM173 Antibody, Biotin conjugated, implement these validation approaches:

• Positive and Negative Controls:

  • Positive Controls: THP-1 and U937 cell lines have been verified to express TMEM173

  • Negative Controls: Consider using cells with TMEM173 knockdown or knockout

  • Genetic Variant Controls: HAQ/HAQ homozygous cells express extremely low TMEM173 protein and can serve as relative negative controls

• Isotype Controls:

  • Use appropriate isotype-matched control antibodies (e.g., IgG for polyclonal antibodies, IgG2a for OTI4H1 clone)

  • Process isotype controls identically to experimental samples

• Multiple Detection Methods:

  • Cross-validate results using different techniques (Western blot, flow cytometry, immunoprecipitation)

  • Compare detection using antibodies targeting different epitopes of TMEM173

• Immunoprecipitation Validation:

  • Perform immunoprecipitation followed by Western blot detection with a different TMEM173 antibody

  • This approach has been used successfully with 2.0 μg of antibody pre-coupled to Dynabeads protein G

• Comparison with Gene Expression Data:

  • Correlate protein detection with mRNA expression levels

  • Consider the genetic background (e.g., HAQ variant) which affects transcript levels

• Blocking Peptide Competition:

  • Pre-incubate antibody with immunizing peptide (e.g., synthetic peptide derived from human TMEM173 immunogen range 231-330/379)

  • Observe reduction in signal intensity to confirm specificity

Implementing these validation strategies provides confidence in the specificity of TMEM173 antibody detection across different experimental applications.

What are the optimal storage conditions to maintain antibody activity?

To maintain optimal activity of TMEM173 Antibody, Biotin conjugated, the following storage conditions are recommended:

• Temperature:

  • Store at -20°C for long-term storage

  • Some preparations may be stored at -80°C for extended stability

  • Avoid repeated freeze-thaw cycles which can degrade antibody activity and biotin conjugation

• Storage Buffer Composition:

  • Typical storage buffer includes:

    • 50% Glycerol to prevent freezing damage

    • 0.01M TBS (pH 7.4) or PBS

    • 1% BSA as a stabilizer

    • 0.03% Proclin300 or 0.09% Sodium Azide as preservatives

• Aliquoting:

  • Upon receipt, divide into small single-use aliquots to minimize freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week if in frequent use

• Handling Precautions:

  • Avoid exposure to light, particularly important for biotin-conjugated antibodies

  • Centrifuge briefly before opening vials to collect solution at the bottom

  • Use sterile techniques when handling to prevent contamination

• Reconstitution (if applicable):

  • For lyophilized antibodies, reconstitute using sterile water or buffer as recommended

  • Allow complete dissolution before aliquoting

When stored properly at -20°C, TMEM173 antibodies typically maintain activity for at least 12 months from the date of receipt .

How can I use TMEM173 Antibody, Biotin conjugated to study genetic variants like HAQ?

Studying TMEM173 genetic variants such as HAQ (R71H-G230A-R293Q) using biotin-conjugated antibodies requires specialized experimental approaches:

• Comparative Expression Analysis:

  • Western blot analysis of TMEM173 expression in cells with known genotypes:

    • R232/R232 (wild-type): Expected normal protein levels

    • R232/HAQ (heterozygous): Expected intermediate protein levels

    • HAQ/HAQ (homozygous): Expected extremely low protein levels

  • Flow cytometry quantification comparing mean fluorescence intensity across genotypes

  • Immunohistochemistry to assess tissue expression patterns in different genetic backgrounds

• Correlation with Functional Responses:

  • Stimulate cells with cyclic dinucleotides (CDNs)

  • Measure downstream signaling responses (phospho-IRF3, phospho-STAT1)

  • Compare interferon production between different genotypes

  • HAQ/HAQ B cells have been documented to show minimal response to CDNs

• Population-Specific Considerations:

  • Include samples from diverse ethnic backgrounds:

    • East Asian populations: ~16.1% homozygous HAQ carriers

    • European populations: ~2.8% homozygous HAQ carriers

    • African populations: Minimal homozygous HAQ representation

• Transcript vs. Protein Analysis:

  • Combine antibody-based protein detection with qPCR for TMEM173 transcript analysis

  • HAQ variant carriers show decreased TMEM173 transcript levels

• Epitope Accessibility Considerations:

  • Ensure the antibody epitope is not affected by the HAQ mutations

  • For TMEM173 Polyclonal Antibody (bs-8335R-Biotin), the immunogen range is 231-330/379, which does not overlap with the HAQ mutations (R71H, G230A, R293Q)

This multi-faceted approach allows for comprehensive analysis of how genetic variants influence TMEM173 expression and function across different experimental systems and population groups.

What techniques can be used to study TMEM173 signaling pathways in primary cells?

Investigating TMEM173 signaling pathways in primary cells requires specialized techniques that maintain physiological relevance while enabling detailed molecular analysis:

• Isolation and Characterization of Primary Cells:

  • Peripheral blood mononuclear cells (PBMCs): Isolated by density gradient centrifugation

  • Cell subset identification: Use lineage markers in combination with TMEM173 staining

  • Flow cytometric detection: 0.25 μg antibody per 10^6 cells for intracellular TMEM173 staining

• Stimulation Protocols:

  • Cyclic dinucleotide (CDN) stimulation: Use cell-permeable CDNs like 2'3'-cGAMP

  • DNA-mediated activation: Transfect primary cells with dsDNA using appropriate methods

  • Monitor kinetics of activation: Examine early (minutes to hours) and late (hours to days) responses

• Multi-parameter Analysis:

  • Phospho-flow cytometry: Measure phosphorylation of downstream targets (IRF3, TBK1, STAT1)

  • Combine with TMEM173 staining to correlate expression with activation status

  • Multiplexed cytokine analysis: Measure IFN-β, IL-6, and other inflammatory cytokines

• Real-time Imaging Approaches:

  • Live-cell imaging of TMEM173 trafficking using fluorescently tagged antibodies or fusion proteins

  • Super-resolution microscopy to examine co-localization with other signaling components

  • FRET-based reporters to detect protein-protein interactions in the pathway

• Single-cell Analysis:

  • Single-cell RNA-seq to examine transcriptional responses downstream of TMEM173 activation

  • Correlation of TMEM173 protein levels (detected by antibody) with single-cell transcriptional profiles

  • CyTOF (mass cytometry) compatible with validated TMEM173 antibodies

• Genetic Background Considerations:

  • Genotype donors for TMEM173 variants (R232, HAQ) to account for expression differences

  • Stratify functional analyses based on genetic background

These approaches allow for detailed characterization of TMEM173 signaling in physiologically relevant primary cells while accounting for genetic and cellular heterogeneity.

How can TMEM173 Antibody be used in studying disease models related to SAVI?

TMEM173 antibodies are valuable tools for investigating STING-associated vasculopathy with onset in infancy (SAVI), an autoinflammatory disease caused by gain-of-function mutations in the TMEM173 gene . Here are methodological approaches for such studies:

• Patient-Derived Cell Analysis:

  • Isolate PBMCs from SAVI patients and healthy controls

  • Compare TMEM173 expression levels and cellular distribution using flow cytometry or immunocytochemistry

  • Recommended antibody concentration: 0.25 μg per 10^6 cells for flow cytometry; 8-25 μg/mL for immunocytochemistry

• Mutation-Specific Studies:

  • Generate cell models expressing SAVI-associated TMEM173 mutations

  • Use immunoprecipitation (2 μg/mL antibody) followed by Western blot to:

    • Assess protein-protein interactions altered by mutations

    • Examine post-translational modifications

  • Compare subcellular localization of wild-type vs. mutant TMEM173

• Functional Assessment:

  • Measure constitutive type I interferon production

  • Assess NF-κB and IRF3 activation states

  • Correlate TMEM173 expression (using antibody detection) with downstream signaling activity

  • Evaluate effects of potential therapeutic compounds on TMEM173 signaling

• Tissue Analysis in Animal Models:

  • Examine TMEM173 expression in affected tissues from SAVI mouse models

  • Perform immunohistochemistry on paraffin-embedded tissues (IHC-P, 1:200-400 dilution) or frozen sections (IHC-F, 1:100-500 dilution)

  • Co-stain with markers of inflammation to correlate TMEM173 activity with pathology

• Biomarker Development:

  • Evaluate TMEM173 as a potential biomarker for disease activity

  • Correlate antibody-detected TMEM173 levels with clinical parameters

  • Develop standardized flow cytometry panels including TMEM173 detection

These approaches enable comprehensive investigation of SAVI pathophysiology and potential therapeutic interventions targeting the TMEM173/STING pathway.

What are the considerations for multiplex detection of TMEM173 with other immune markers?

Multiplex detection of TMEM173 with other immune markers provides valuable insights into the contextual role of STING in immune responses. Here are key considerations for developing effective multiplex panels:

• Panel Design Strategy:

  • Combine TMEM173 with markers of:

    • Cell lineage (CD3, CD19, CD14, CD11c)

    • Activation status (CD69, CD25, HLA-DR)

    • Related signaling molecules (TBK1, IRF3, STAT1)

  • Consider cellular localization when selecting markers (membrane, cytoplasmic, nuclear)

• Technical Considerations for Biotin-Conjugated TMEM173 Antibody:

  • Avoid other biotin-conjugated antibodies in the panel to prevent cross-reactivity

  • Use streptavidin conjugated to a unique fluorophore not overlapping with other markers

  • If using fluorophore-labeled streptavidin, add it in a separate step after all other antibodies

• Fixation and Permeabilization Optimization:

  • Balance requirements for intracellular TMEM173 detection with preservation of other markers

  • Paraformaldehyde fixation followed by saponin permeabilization works well for TMEM173

  • Consider sequential staining protocols:

    1. Stain surface markers

    2. Fix and permeabilize

    3. Stain intracellular markers including TMEM173

• Spectral Considerations:

  • For flow cytometry: Place TMEM173 detection in bright channels if expression is low

  • For imaging: Select fluorophores with minimal spectral overlap

  • Include appropriate controls for spectral compensation

• Validation Requirements:

  • Single-stain controls for each marker

  • Fluorescence-minus-one (FMO) controls to set accurate gates

  • Isotype controls to assess non-specific binding

  • Biological controls with known expression patterns

• Data Analysis Approaches:

  • Use dimensionality reduction techniques (tSNE, UMAP) to visualize relationships

  • Apply clustering algorithms to identify cell populations with co-expression patterns

  • Correlate TMEM173 expression with functional readouts

By carefully addressing these considerations, researchers can effectively incorporate TMEM173 Antibody, Biotin conjugated into multiplex panels for comprehensive immune phenotyping.

Why might I observe variable TMEM173 expression levels across different samples?

Variability in TMEM173 expression can stem from multiple factors that must be considered during experimental design and data interpretation:

• Genetic Variation:

  • The human TMEM173 gene exhibits significant heterogeneity with population stratification

  • The HAQ (R71H-G230A-R293Q) allele carriers show extremely low TMEM173 protein expression

  • Homozygous HAQ individuals account for ~16.1% of East Asians and ~2.8% of Europeans

  • Genotyping samples for TMEM173 variants can explain expression differences

• Cell Type-Specific Expression:

  • TMEM173 expression varies across cell types

  • Consistently detectable in THP-1 and U937 human cell lines

  • Expression levels differ between monocytes, macrophages, dendritic cells, and lymphocytes

  • Consider baseline expression profiles when comparing across cell types

• Activation State:

  • TMEM173 expression can be modulated by cellular activation

  • Stimulation with TLR ligands, interferons, or other inflammatory mediators may alter expression

  • Document treatment conditions and activation markers when reporting TMEM173 levels

• Technical Factors:

  • Antibody concentration: Ensure consistent application (e.g., 0.2 μg/mL for Western blot)

  • Fixation/permeabilization: Incomplete permeabilization reduces detection of intracellular epitopes

  • Signal-to-noise ratio: Background from non-specific binding can mask true expression differences

  • Sample processing: Time between collection and analysis can affect protein stability

• Analytical Considerations:

  • Flow cytometry: Use appropriate controls to set positive/negative boundaries

  • Western blot: Include loading controls (β-actin, GAPDH) to normalize for total protein

  • Quantification method: Mean fluorescence intensity vs. percent positive cells can yield different results

Understanding these sources of variability allows for proper experimental design with appropriate controls and accurate interpretation of TMEM173 expression data across different experimental conditions and sample types.

How can I address non-specific binding when using TMEM173 Antibody, Biotin conjugated?

Non-specific binding can compromise the reliability of TMEM173 detection. Here are methodological approaches to minimize this issue:

• Optimized Blocking Protocols:

  • Use effective blocking agents:

    • 1-5% BSA in PBS or TBS

    • 5-10% normal serum from the same species as the secondary antibody

    • Commercial blocking buffers optimized for biotin-conjugated antibodies

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C

  • Include blocking agent in antibody dilution buffer

• Titration of Antibody Concentration:

  • Perform antibody titration experiments to identify optimal concentration

  • For Western blot: Start with recommended 0.2 μg/mL and adjust as needed

  • For immunocytochemistry: Test range around recommended 8-25 μg/mL

  • For flow cytometry: Compare signal-to-noise ratio at various concentrations around 0.25 μg per 10^6 cells

• Validation with Controls:

  • Include isotype controls (e.g., IgG for polyclonal, IgG2a for monoclonal antibodies)

  • Use known negative cells (consider HAQ/HAQ genotype cells with minimal TMEM173 expression)

  • Perform antibody pre-absorption with immunizing peptide

• Reduction of Endogenous Biotin Interference:

  • Include avidin/biotin blocking step if using biotin-rich tissues

  • Use commercial avidin/biotin blocking kits before applying biotin-conjugated antibodies

  • Consider streptavidin/biotin blocking systems for tissues with high endogenous biotin

• Optimization of Wash Steps:

  • Increase number of washes after antibody incubation

  • Use detergent-containing wash buffers (0.05-0.1% Tween-20)

  • Extend wash times to effectively remove unbound antibody

• Secondary Detection Optimization:

  • For biotin-conjugated antibodies, titrate streptavidin-conjugate concentration

  • Use highly cross-adsorbed streptavidin reagents

  • Consider fluorophore or enzyme selection based on autofluorescence or endogenous enzyme activity

By systematically implementing these approaches, researchers can significantly reduce non-specific binding and improve the specificity of TMEM173 detection in various experimental systems.

How should I interpret contradictory findings between different detection methods?

When faced with contradictory results between different methods of TMEM173 detection, a systematic approach to reconciliation is required:

• Method-Specific Considerations:

  Detection Method	Common Issues	Resolution Strategies
  Western Blot	- Band size discrepancies - Multiple bands - Weak signal	- Verify reducing conditions - Test different lysis buffers - Confirm molecular weight (37-40 kDa)
  Flow Cytometry	- Low signal-to-noise ratio - Autofluorescence - Permeabilization variability	- Optimize fixation/permeabilization - Use validated protocols with paraformaldehyde/saponin - Include FMO controls
  IHC/ICC	- Background staining - Weak specific signal - Epitope masking	- Adjust antibody concentration (8-25 μg/mL) - Optimize antigen retrieval - Test different blocking methods
  ELISA	- Matrix effects - Hook effect at high concentrations	- Perform dilution series - Use recommended dilution range (1:500-1000)

• Epitope Accessibility Variations:

  • Different methods expose different epitopes

  • Native vs. denatured protein conformation affects antibody binding

  • For TMEM173 Polyclonal Antibody (bs-8335R-Biotin), the immunogen range (231-330/379) may be differently accessible in various applications

• Expression Level Threshold Detection:

  • Methods vary in sensitivity

  • Western blot may detect aggregate protein in a sample while flow cytometry reports per-cell expression

  • Consider absolute detection limits of each method

• Reconciliation Strategies:

  • Biological validation: Correlate results with functional readouts

  • Orthogonal approaches: Use alternative antibodies targeting different epitopes

  • Genetic validation: Test in systems with manipulated TMEM173 expression (overexpression, knockdown)

  • Cross-validation table:

  Finding	Western Blot	Flow Cytometry	Interpretation
  Positive	Positive	Consistent results confirm expression
  Positive	Negative	Potential aggregate detection in Western blot or insufficient permeabilization in flow cytometry
  Negative	Positive	Possible antibody specificity issue or sample processing differences
  Negative	Negative	Consistent results suggest absence of expression

• Documentation and Reporting:

  • Report detailed methodologies for each technique

  • Present all data transparently, including contradictions

  • Discuss potential methodological limitations

  • Consider genetic background (e.g., HAQ variant) as potential explanation for discrepancies

What experimental controls are essential when studying TMEM173 function and expression?

Robust experimental design for TMEM173 studies requires comprehensive controls to ensure reliable data interpretation:

• Antibody Validation Controls:

  • Isotype Controls: Matched to the TMEM173 antibody class (e.g., IgG for polyclonal, IgG2a for monoclonal OTI4H1)

  • Peptide Competition: Pre-incubation with immunizing peptide should abolish specific signal

  • Genetic Controls: Cells with TMEM173 knockdown/knockout or HAQ/HAQ genotype with minimal expression

  • Cross-Reactivity Assessment: Test antibody on cells from different species if claiming cross-reactivity

• Expression Analysis Controls:

  • Positive Expression Controls: THP-1 and U937 human cell lines show reliable TMEM173 expression

  • Loading Controls: β-actin, GAPDH, or total protein stains for Western blot normalization

  • Technical Replicates: Minimum of three independent experiments to assess reproducibility

  • Biological Replicates: Samples from multiple donors to account for genetic variability

• Functional Assay Controls:

  • Positive Stimulation Controls: Known STING agonists (e.g., 2'3'-cGAMP, DMXAA for mouse)

  • Negative Controls: Structurally similar but inactive compounds

  • Pathway Inhibition: TBK1 inhibitors (e.g., BX795) to confirm STING-dependent signaling

  • Genotype Controls: Compare wild-type (R232/R232) with HAQ variant carriers

• Genetic Background Considerations:

  • Document TMEM173 genotype of cell lines and primary cells

  • Include cells with different TMEM173 variants when possible:

    • R232/R232: Standard expression and function

    • R232/HAQ: Intermediate expression

    • HAQ/HAQ: Minimal expression and function

• Data Acquisition Controls:

  • For Flow Cytometry:

    • Unstained controls

    • Single-stain compensation controls

    • Fluorescence-minus-one (FMO) controls

    • Consistent instrument settings across experiments

  • For Western Blot:

    • Molecular weight markers

    • Concentration gradients to confirm linearity of detection

    • Consistent exposure parameters for quantitative comparisons

Implementing these controls ensures that experimental findings related to TMEM173 can be interpreted with confidence and facilitates comparison of results across different studies and laboratories.