TNFRSF14 Human, Sf9

What is TNFRSF14 Human, Sf9 and how is it produced?

TNFRSF14 Human, Sf9 refers to recombinant human TNFRSF14 protein expressed in Spodoptera frugiperda 9 (Sf9) insect cells using a baculovirus expression system. It is typically produced as a single, glycosylated polypeptide chain containing 406 amino acids (residues 39-202 of the native sequence) with a molecular mass of approximately 46.6kDa. The recombinant protein is often fused to tags such as a C-terminal IgG His-Tag to facilitate purification through proprietary chromatographic techniques. This expression system is preferred for complex mammalian proteins as it allows proper protein folding and post-translational modifications, particularly glycosylation, which are crucial for maintaining the protein's biological functions .

What are the common synonyms and designations for TNFRSF14?

TNFRSF14 is known by multiple names in scientific literature, which can sometimes create confusion when searching databases. Common synonyms include: Tumor necrosis factor receptor superfamily member 14 isoform 1, ATAR, CD270, HVEA, HVEM, LIGHTR, TR2, Herpes virus entry mediator A, and Herpesvirus entry mediator A. Understanding these alternative designations is essential for comprehensive literature searches when conducting research on this protein. When citing research, it's advisable to include the primary designation (TNFRSF14) along with the most commonly used alternative (HVEM) to ensure clarity across different research fields .

What are the key structural features of recombinant TNFRSF14?

Recombinant TNFRSF14 produced in Sf9 cells is a glycosylated type I transmembrane protein. The commercial recombinant form typically contains amino acids 39-202, which represents the extracellular domain responsible for ligand binding. When expressed with a C-terminal IgG His-Tag fusion (adding 239 amino acids), the total molecular weight is approximately 46.6kDa, though it may appear as 40-57kDa on SDS-PAGE due to glycosylation patterns. The protein's functional domains include cysteine-rich regions characteristic of TNF receptor family members, which are critical for the formation of hexameric complexes with its ligands. These structural features are essential considerations when designing experiments involving protein-protein interactions or receptor signaling studies .

What are the primary research applications for TNFRSF14 Human, Sf9?

TNFRSF14 Human, Sf9 is utilized in multiple research contexts including:

• Receptor-ligand binding studies: Investigating interactions with ligands such as TNFSF14/LIGHT, LTA/lymphotoxin-alpha, BTLA, and CD160.

• Immunological signaling research: Examining co-stimulatory pathways in T-cell activation and inhibitory signaling networks.

• Flow cytometry analysis: Detecting HVEM expression on various immune cell populations including T cells, B cells, and NK cells.

• Viral pathogenesis studies: Understanding TNFRSF14's role as an entry receptor for herpes simplex viruses.

• Development of immunotherapeutics: Screening potential modulators of the HVEM signaling network for therapeutic applications.

These applications require different experimental approaches and optimization strategies depending on the specific research questions being addressed .

How can I optimize flow cytometry protocols for detecting TNFRSF14 expression?

Optimizing flow cytometry protocols for TNFRSF14 detection requires careful consideration of several parameters:

• Antibody selection: Use validated anti-HVEM/TNFRSF14 monoclonal antibodies (such as MAB3563) with demonstrated specificity.

• Cell preparation: When working with PBMCs, proper gating strategies are essential. Gate on lymphocyte populations using markers such as CD14 (negative), CD3 (for T cells), CD19 (for B cells), and CD56 (for NK cells).

• Fluorophore selection: Choose appropriate fluorophore combinations that minimize spectral overlap. For example, using Allophycocyanin-conjugated secondary antibodies for TNFRSF14 detection while using other fluorophores (PE, Alexa Fluor® 488, Alexa Fluor® 700) for lineage markers.

• Membrane protein staining considerations: Follow specialized protocols for membrane-associated proteins to preserve epitope accessibility. This may include gentle fixation methods and specialized buffers.

• Controls: Always include isotype controls (e.g., MAB1050) and single-color controls for proper compensation.

This approach allows for reliable detection of TNFRSF14 across different immune cell populations in complex samples .

What experimental considerations should be taken when studying TNFRSF14-ligand interactions?

When studying TNFRSF14-ligand interactions, researchers should consider:

• Complex formation dynamics: The interaction of TNFSF14/LIGHT with TNFRSF14/HVEM forms hexameric complexes (3 ligand molecules with 3 receptor molecules), which influences experimental design for binding assays.

• Multiple ligand partners: TNFRSF14 binds to both TNF superfamily members (TNFSF14/LIGHT and LTA/lymphotoxin-alpha) and immunoglobulin superfamily members (BTLA and CD160), potentially creating competitive binding scenarios.

• Cis versus trans interactions: TNFRSF14 can interact with its ligands in cis (on the same cell) or trans (between different cells) configurations, which significantly impacts the biological outcome of these interactions.

• Physiological relevance: Using physiologically relevant concentrations of both receptor and ligands, as non-physiological concentrations may lead to artifactual binding patterns.

• Detection methods: Employing multiple complementary methods (ELISA, surface plasmon resonance, co-immunoprecipitation) to validate binding interactions and determine kinetic parameters.

These considerations are crucial for accurately interpreting the complex signaling network mediated by TNFRSF14 .

What are the optimal storage and handling conditions for TNFRSF14 Human, Sf9?

To maintain optimal activity of TNFRSF14 Human, Sf9 recombinant protein:

• Short-term storage (2-4 weeks): Store at 4°C in the original buffer formulation.

• Long-term storage: Store at -20°C with added carrier protein (0.1% HSA or BSA) to prevent protein adsorption and maintain stability.

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces protein activity. Prepare appropriate aliquots before freezing.

• Buffer considerations: The protein is typically supplied in phosphate-buffered saline (pH 7.4) with 10% glycerol. This formulation helps maintain protein stability during storage.

• Working conditions: When using the protein for experiments, maintain it on ice and use within the same day after thawing to ensure maximum activity.

Following these guidelines will help maintain protein quality and experimental reproducibility .

How can I assess the quality and functional activity of TNFRSF14 preparations?

Assessing quality and functional activity of TNFRSF14 preparations involves several complementary approaches:

• Purity assessment:

  • SDS-PAGE analysis to confirm >90% purity

  • Western blotting with specific anti-TNFRSF14 antibodies

  • Size exclusion chromatography to detect aggregation

• Structural integrity:

  • Circular dichroism spectroscopy to evaluate secondary structure

  • Limited proteolysis to assess proper folding

• Functional activity:

  • Binding assays with known ligands (TNFSF14/LIGHT, BTLA)

  • Cell-based reporter assays measuring downstream signaling activation

  • Competitive binding assays with validated TNFRSF14 preparations

• Glycosylation analysis:

  • Lectin binding assays or mass spectrometry to characterize glycan profiles

Each batch should be tested against reference standards to ensure consistent quality and activity before use in critical experiments .

What methods can be used to study TNFRSF14-mediated signaling pathways?

To investigate TNFRSF14-mediated signaling pathways, researchers can employ:

• Phosphorylation studies:

  • Western blotting to detect phosphorylation of downstream signaling molecules

  • Phospho-specific flow cytometry for single-cell resolution of signaling events

  • Phospho-proteomics for global analysis of signaling networks

• Gene expression analysis:

  • qRT-PCR for targeted gene expression studies

  • RNA-seq for genome-wide transcriptional responses

  • NF-κB reporter assays to measure pathway activation

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify signaling complexes

  • Proximity ligation assays for in situ detection of protein interactions

  • FRET/BRET approaches for real-time interaction monitoring

• Functional cellular assays:

  • T cell proliferation assays using CFSE dilution

  • Cytokine production measurement (IFN-γ, GM-CSF)

  • Cell survival/apoptosis assays following TNFRSF14 engagement

These methodologies can reveal the complex signaling networks activated by TNFRSF14 engagement under different conditions and with different binding partners .

How does the glycosylation profile of Sf9-expressed TNFRSF14 differ from mammalian-expressed protein, and what are the functional implications?

The glycosylation profile of Sf9-expressed TNFRSF14 differs significantly from mammalian-expressed protein due to fundamental differences in insect and mammalian glycosylation machinery:

• Structural differences:

  • Sf9-expressed proteins contain high-mannose type N-glycans without complex branching

  • Lack terminal sialylation commonly found in mammalian glycoproteins

  • Absence of certain mammalian-specific glycan structures

• Functional implications:

  • Altered receptor-ligand binding kinetics and affinity may occur

  • Different protein half-life in experimental systems

  • Potentially modified interaction with lectins or glycan-binding proteins

• Experimental considerations:

  • For binding studies focusing on protein-protein interfaces not affected by glycosylation, Sf9-expressed protein may be adequate

  • For studies where glycosylation impacts function (e.g., in vivo studies), mammalian expression systems might be preferable

  • Comparative studies using both expression systems can help distinguish glycan-dependent and independent functions

Researchers should consider these differences when designing experiments and interpreting results, especially when translating findings to mammalian systems .

What are the challenges in studying the dual stimulatory and inhibitory functions of TNFRSF14 in immune regulation?

Investigating the dual stimulatory and inhibitory functions of TNFRSF14 presents several challenges:

• Context-dependent signaling:

  • TNFRSF14 delivers costimulatory signals when binding TNFSF14/LIGHT, promoting T cell proliferation and IFN-γ production

  • Inhibitory signaling occurs when binding BTLA, suppressing immune responses

  • These opposing outcomes complicate experimental design and interpretation

• Cis vs. trans interactions:

  • TNFRSF14 can interact with BTLA on the same cell (cis) or on different cells (trans)

  • Cis interactions appear to regulate immune responses in naive T cells

  • Trans interactions predominate during adaptive immune responses

  • Distinguishing these interactions experimentally requires sophisticated approaches

• Cell type specificity:

  • Effects vary significantly between T cells, B cells, NK cells, and non-immune cells

  • Specialized experimental systems may be needed for each cell type

• Compensatory mechanisms:

  • Redundancy in signaling pathways can mask phenotypes in experimental systems

  • Multiple ligands competing for the same receptor complicate interpretation

Addressing these challenges requires carefully designed experiments with appropriate controls, combined with systems biology approaches to decipher the complex signaling networks .

How can we reconcile contradictory findings on TNFRSF14 function in different experimental systems?

Contradictory findings regarding TNFRSF14 function can be reconciled through systematic analysis of experimental variables:

• Expression level considerations:

  • Overexpression systems may alter the balance of signaling pathways

  • Physiological versus non-physiological expression levels can lead to different outcomes

  • Quantitative analysis of receptor density should be included in experimental reporting

• Ligand competition effects:

  • The presence of multiple ligands (LIGHT, LTA, BTLA, CD160) can create competitive binding scenarios

  • Relative concentrations of each ligand may determine net signaling outcome

  • Single ligand studies may not reflect the complexity of in vivo settings

• Cell type and activation state:

  • TNFRSF14 functions differently in naive versus memory T cells

  • Effects vary between T cells, B cells, NK cells, and epithelial cells

  • Activation state alters receptor distribution and signaling partnerships

• Experimental timeframes:

  • Short-term versus long-term signaling outcomes may differ substantially

  • Acute versus chronic engagement of the receptor leads to different cellular responses

• Integration of multiple readouts:

  • Combining molecular (signaling), cellular (functional), and systems-level (in vivo) readouts

  • Using complementary techniques to validate findings across different experimental platforms

This comprehensive approach can help resolve apparent contradictions and develop a unified model of TNFRSF14 function in immune regulation .

Why might recombinant TNFRSF14 show reduced binding activity after storage, and how can this be prevented?

Recombinant TNFRSF14 may experience reduced binding activity during storage due to several factors:

• Protein aggregation:

  • Formation of aggregates reduces the effective concentration of properly folded protein

  • Solution: Add carrier proteins (0.1% HSA or BSA) to prevent protein-protein interactions

  • Use size exclusion chromatography or dynamic light scattering to monitor aggregation state

• Oxidation of cysteine residues:

  • TNFRSF14 contains critical cysteine residues that form disulfide bonds

  • Oxidation can disrupt protein structure and function

  • Solution: Add reducing agents like DTT or β-mercaptoethanol (at low concentrations) to storage buffers

• Proteolytic degradation:

  • Even trace amounts of proteases can degrade the protein during storage

  • Solution: Add protease inhibitors to storage buffers

  • Store at -80°C for very long-term preservation

• Adsorption to container surfaces:

  • Protein loss through binding to storage tube surfaces

  • Solution: Use low-binding tubes and add carrier proteins

• Freeze-thaw damage:

  • Repeated freeze-thaw cycles can damage protein structure

  • Solution: Prepare single-use aliquots before freezing

• Glycan modifications:

  • Gradual alteration of glycan structures during storage

  • Solution: Validate protein activity after extended storage periods

Implementation of these preventive measures can significantly extend the functional shelf-life of recombinant TNFRSF14 preparations .

What are common pitfalls in experimental design when studying TNFRSF14-mediated immune responses?

Common pitfalls in TNFRSF14 research include:

• Insufficient characterization of cellular models:

  • Failure to quantify baseline expression of TNFRSF14 and its ligands

  • Solution: Perform comprehensive flow cytometry or western blot analysis before experiments

• Overlooking competing receptor-ligand interactions:

  • TNFRSF14 interactions with multiple ligands create complex signaling networks

  • Solution: Consider using blocking antibodies or genetic approaches to isolate specific interactions

• Inadequate controls for recombinant proteins:

  • Protein preparations may contain contaminants affecting results

  • Solution: Include appropriate controls (heat-inactivated protein, irrelevant proteins) in experimental design

• Neglecting the impact of tags on protein function:

  • His-tags or Fc-fusions may alter binding properties or induce signaling

  • Solution: Compare tagged and untagged versions or use different tag positions

• Misinterpreting cell death phenotypes:

  • TNFRSF14 can promote both survival and apoptosis depending on context

  • Solution: Use multiple assays to distinguish different modes of cell death (apoptosis, necroptosis)

• Overgeneralizing findings across cell types:

  • Effects in one immune cell type may not extend to others

  • Solution: Validate findings across relevant cell populations

• Neglecting species-specific differences:

  • Human and murine TNFRSF14 systems have important functional differences

  • Solution: Acknowledge limitations when translating between species models

Avoiding these pitfalls requires careful experimental planning and appropriate controls .

How can researchers optimize co-immunoprecipitation protocols for studying TNFRSF14 protein interactions?

Optimizing co-immunoprecipitation (co-IP) protocols for TNFRSF14 interactions requires:

• Lysis buffer optimization:

  • Use mild detergents (0.5-1% NP-40, CHAPS, or digitonin) to preserve protein interactions

  • Include protease and phosphatase inhibitors to prevent degradation

  • Test multiple buffer conditions to identify optimal composition for specific interactions

• Crosslinking considerations:

  • For transient interactions, consider mild crosslinking (0.5-2% formaldehyde)

  • Optimize crosslinking time and concentration to prevent over-crosslinking

  • Include controls to assess crosslinking efficiency

• Antibody selection strategy:

  • Test multiple antibodies targeting different epitopes of TNFRSF14

  • Validate antibody specificity using knockout/knockdown controls

  • Consider using tagged versions (His-tag, FLAG-tag) for reliable pull-down

• Preclearing procedures:

  • Implement preclearing step with protein A/G beads to reduce non-specific binding

  • Include isotype control antibodies to identify non-specific interactions

• Washing optimization:

  • Balance between stringency (to reduce background) and preserving interactions

  • Test wash buffers with different salt concentrations and detergent levels

  • Perform multiple short washes rather than fewer long washes

• Detection strategies:

  • For known interactions: western blotting with specific antibodies

  • For discovery of novel interactions: mass spectrometry analysis

  • Consider proximity-based labeling methods (BioID, APEX) as complementary approaches

These optimizations can significantly improve the detection of physiologically relevant TNFRSF14 protein interactions while minimizing artifacts .

How might structural biology approaches advance our understanding of TNFRSF14 signaling complexes?

Structural biology approaches offer significant potential to advance TNFRSF14 research:

• Cryo-electron microscopy (cryo-EM):

  • Can resolve the hexameric complexes formed between TNFRSF14 and its ligands

  • Allows visualization of conformational changes upon ligand binding

  • May reveal the structural basis for differential signaling outcomes with different ligands

• X-ray crystallography:

  • Higher resolution studies of specific domain interactions

  • Co-crystal structures with different binding partners (LIGHT, BTLA, CD160)

  • Structure-guided design of selective modulators

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Can map binding interfaces and conformational changes in solution

  • Particularly useful for studying dynamic aspects of receptor-ligand interactions

  • Requires less protein than crystallography and works with more challenging protein complexes

• Single-molecule studies:

  • FRET-based approaches to monitor binding events in real-time

  • Can capture transient intermediates in complex formation

  • Allows study of binding kinetics under near-physiological conditions

• Molecular dynamics simulations:

  • Integrate experimental structural data with computational approaches

  • Predict conformational changes and binding energetics

  • Model the impact of mutations or post-translational modifications

These approaches could resolve fundamental questions regarding the structural basis of TNFRSF14's dual stimulatory and inhibitory functions .

What are promising therapeutic applications targeting the TNFRSF14 signaling axis in immune disorders?

The TNFRSF14 signaling axis presents several promising therapeutic targets:

• Autoimmune disorders:

  • Selective modulation of TNFRSF14-BTLA inhibitory interactions to dampen pathological immune responses

  • Development of agonistic antibodies promoting inhibitory signaling

  • Targeting TNFRSF14-LIGHT stimulatory interactions to reduce inflammatory cascades

• Cancer immunotherapy:

  • Enhancing TNFRSF14-LIGHT interactions to promote anti-tumor immune responses

  • Blocking inhibitory BTLA interactions to enhance T cell activity against tumors

  • Combination approaches with checkpoint inhibitors targeting complementary pathways

• Infectious disease:

  • Modulating TNFRSF14 to enhance immunity against chronic viral infections

  • Exploiting the role of TNFRSF14 as a herpesvirus entry mediator for antiviral strategies

  • Targeting epithelial TNFRSF14 signaling to enhance antimicrobial protein production

• Transplantation:

  • Promoting TNFRSF14-mediated inhibitory signaling to prevent graft rejection

  • Developing targeted approaches to modulate specific lymphocyte subsets involved in rejection

• Biologics development approaches:

  • Bi-specific antibodies targeting specific TNFRSF14 interactions

  • Engineered ligands with modified binding properties

  • Decoy receptors that selectively block specific interactions

These therapeutic directions require careful consideration of the complex and sometimes opposing functions of TNFRSF14 in different cellular contexts .

How can single-cell technologies enhance our understanding of TNFRSF14 function in heterogeneous immune cell populations?

Single-cell technologies offer powerful approaches for dissecting TNFRSF14 function:

• Single-cell RNA sequencing (scRNA-seq):

  • Reveals heterogeneity in TNFRSF14 and ligand expression across immune populations

  • Identifies cell subsets with unique receptor expression patterns

  • Maps transcriptional consequences of receptor engagement at single-cell resolution

• Single-cell proteomics (mass cytometry/CyTOF):

  • Simultaneously measures surface TNFRSF14 expression and intracellular signaling

  • Captures pathway activation with dozens of parameters per cell

  • Allows identification of rare cell populations with unique receptor activity

• Single-cell signaling analysis:

  • Phospho-flow cytometry to track signaling events in individual cells

  • Reveals how signaling varies across cell types and activation states

  • Identifies threshold effects in receptor signaling

• Spatial transcriptomics/proteomics:

  • Maps TNFRSF14 expression and activity within tissue microenvironments

  • Reveals spatial relationships between receptor-expressing and ligand-expressing cells

  • Provides context for understanding in vivo functions

• Live cell imaging techniques:

  • Single-molecule tracking to visualize receptor dynamics and clustering

  • Real-time monitoring of signaling events in individual cells

  • Captures temporal aspects of receptor engagement and downstream responses

These technologies can resolve conflicting reports on TNFRSF14 function by revealing how receptor activity varies across cell types, activation states, and tissue contexts .

What are the key considerations when using TNFRSF14 Human, Sf9 in receptor-ligand binding assays?

When designing receptor-ligand binding assays with TNFRSF14 Human, Sf9, researchers should consider:

• Assay format selection:

  Format	Advantages	Limitations	Best For
  ELISA	High-throughput, quantitative	Limited information on kinetics	Screening studies
  Surface Plasmon Resonance	Real-time kinetics, label-free	Requires specialized equipment	Detailed binding parameters
  Bio-Layer Interferometry	Real-time kinetics, less sample	Lower sensitivity than SPR	Rapid kinetic screening
  AlphaScreen/AlphaLISA	High sensitivity, homogeneous	Potential light interference	High-throughput screening

• Protein immobilization strategy:

  • Direct coating may alter protein conformation

  • Consider oriented immobilization using the His-tag

  • Test multiple capture approaches to ensure optimal ligand binding

• Buffer optimization:

  • Divalent cations (Ca²⁺, Mg²⁺) may influence binding

  • Test different pH conditions (typically pH 6.8-7.8)

  • Include appropriate blocking agents to minimize background

• Control experiments:

  • Include known binding partners as positive controls

  • Use irrelevant proteins as negative controls

  • Perform competition assays to confirm binding specificity

• Data analysis considerations:

  • Account for potential avidity effects in multimeric interactions

  • Consider cooperative binding models for data fitting

  • Report both kinetic and equilibrium binding parameters when possible

These considerations will help ensure reliable and reproducible binding data when working with TNFRSF14 Human, Sf9 .

How should researchers approach experimental design for studying TNFRSF14 in viral entry mechanisms?

When investigating TNFRSF14/HVEM in viral entry mechanisms, particularly for herpes simplex viruses, researchers should:

• Model system selection:

  • Cell lines with defined TNFRSF14 expression levels

  • Primary cells that naturally express TNFRSF14

  • Reconstitution systems in TNFRSF14-negative cells

• Viral entry assay considerations:

  • Distinguish binding from fusion/entry events

  • Use reporter viruses for quantitative measurements

  • Consider time-course experiments to capture entry kinetics

• Competitive inhibition approaches:

  • Recombinant TNFRSF14-Fc fusion proteins as competitive inhibitors

  • Anti-TNFRSF14 blocking antibodies

  • Soluble viral glycoprotein D (gD) competitors

• Domain mapping studies:

  • Site-directed mutagenesis of key TNFRSF14 residues

  • Chimeric receptors to identify critical binding regions

  • Truncation mutants to define minimal binding domains

• Visualization techniques:

  • Fluorescently labeled viruses for tracking entry

  • Confocal microscopy to monitor co-localization with endocytic markers

  • Super-resolution microscopy for detailed receptor clustering analysis

• Alternative receptor considerations:

  • Account for redundant entry mechanisms through nectin-1 and other receptors

  • Design experiments to isolate TNFRSF14-specific entry events

  • Compare entry efficiency across cell types with different receptor expression profiles

These approaches provide a comprehensive framework for investigating the complex role of TNFRSF14 in viral pathogenesis .

What methodological approaches are most effective for studying the TNFRSF14-BTLA inhibitory signaling axis?

To effectively study the TNFRSF14-BTLA inhibitory signaling axis, researchers should implement:

• Cell system considerations:

  • Primary T cells expressing physiological levels of both receptors

  • Reconstitution systems in cell lines for controlled expression

  • In vivo models with conditional deletion or expression

• Distinguishing cis vs. trans interactions:

  • Co-expression systems to study cis (same-cell) interactions

  • Co-culture experiments for trans (different-cell) interactions

  • Proximity ligation assays to visualize interactions in situ

• Functional readouts:

  • T cell proliferation assays (CFSE dilution)

  • Cytokine production measurement (ELISA, intracellular cytokine staining)

  • Calcium flux assays for early signaling events

  • Phosphorylation of signaling intermediates (SHP-1/2, ITIM motifs)

• Temporal considerations:

  • Acute vs. chronic receptor engagement

  • Pre-activation vs. simultaneous engagement with TCR signaling

  • Memory formation and recall response effects

• Molecular tools:

  • Structure-guided mutations disrupting specific interactions

  • Domain-specific blocking antibodies

  • Engineered ligands with altered binding properties

• In vivo validation:

  • Adoptive transfer experiments with modified T cells

  • Disease models where inhibitory signaling is relevant (autoimmunity, infection)

  • Comparative analysis across different tissue microenvironments

These methodological approaches enable comprehensive analysis of this complex inhibitory signaling axis across different biological contexts and activation states .

What are the key technical specifications of commercially available TNFRSF14 Human, Sf9?

The following technical specifications are typical for commercially available TNFRSF14 Human, Sf9 preparations:

These specifications provide a reference point for researchers when selecting commercial reagents or producing recombinant protein in-house. Actual values may vary between different suppliers and batches .

What amino acid sequence and structural regions are most critical for TNFRSF14 function?

The critical regions and residues in TNFRSF14 for its various functions include:

• Cysteine-rich domains (CRDs):

  • Characteristic of TNF receptor superfamily

  • Form disulfide bonds essential for proper protein folding

  • Key sequence includes cysteine pattern: CKEDEDYPVGSECCPKCSPGYRVKEACGELTGTVCEP

• Ligand binding interfaces:

  • LIGHT/TNFSF14 binding region: primarily involves CRD2 and CRD3

  • BTLA binding region: distinct from LIGHT binding site, involves CRD1

  • HSV gD binding: overlaps with BTLA binding site

• Transmembrane domain:

  • Residues 202-222 (not present in soluble recombinant forms)

  • Important for receptor clustering and signaling

• Intracellular signaling domain:

  • Contains TRAF binding motifs for signal transduction

  • Mediates interaction with the TRAF2-TRAF3 E3 ligase pathway

  • Critical for downstream activation of NF-κB and other signaling pathways

• Glycosylation sites:

  • Multiple N-linked glycosylation sites in the extracellular domain

  • Influence protein folding, stability, and potentially ligand binding

Understanding these critical regions is essential for designing targeted mutations, developing blocking agents, and interpreting the functional impact of naturally occurring polymorphisms .

What are common quality control tests to validate TNFRSF14 Human, Sf9 before experimental use?

Before using TNFRSF14 Human, Sf9 in experiments, researchers should perform these quality control tests:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (should show >90% purity)

  • Western blot with anti-TNFRSF14 and anti-tag antibodies

  • Mass spectrometry to confirm identity and detect contaminants

• Structural integrity:

  • Circular dichroism to verify secondary structure elements

  • Thermal shift assays to assess protein stability

  • Native PAGE to detect aggregation or oligomerization

• Functional validation:

  • ELISA-based binding assays with known ligands (LIGHT/TNFSF14)

  • Surface plasmon resonance to confirm binding kinetics

  • Cell-based reporter assays measuring functional activity

• Endotoxin testing:

  • LAL (Limulus Amebocyte Lysate) assay

  • Endotoxin levels should be <1.0 EU/μg protein

• Glycosylation analysis:

  • PNGase F treatment to confirm presence of N-linked glycans

  • Lectin blotting to characterize glycan structures

  • Mass spectrometry for detailed glycan profiling

• Batch-to-batch comparison:

  • Activity comparison with previous validated batches

  • Consistent binding parameters with reference ligands

These quality control measures ensure experimental reproducibility and reliable interpretation of results when working with TNFRSF14 preparations .