STYX Human (26-223)

What is STYX Human (26-223) recombinant protein and what distinguishes it from other phosphatases?

STYX Human (26-223) recombinant protein is a single polypeptide chain containing 221 amino acids spanning positions 26-223 of the native STYX protein, with a molecular mass of approximately 25.0 kDa . The commercially available recombinant form typically includes a 23 amino acid His-tag at the N-terminus and is produced in E. coli expression systems .

What distinguishes STYX from conventional phosphatases is its classification as a pseudophosphatase. Unlike active phosphatases, STYX contains a glycine residue instead of the conserved cysteine residue in the dual-specificity phosphatase (dsPTPase) catalytic loop . This substitution renders it catalytically inactive while preserving its ability to bind phosphorylated substrates . This unique property suggests that STYX may function as a regulatory molecule by protecting specific phosphorylated substrates from active phosphatases in cellular pathways .

What are the optimal storage and handling conditions for STYX Human (26-223) recombinant protein?

Proper storage and handling of STYX Human (26-223) recombinant protein are critical for maintaining its structural integrity and functional properties in research applications. The recommended storage conditions are:

For long-term storage, it is recommended to add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) to prevent protein loss through adsorption to storage vessels . Multiple freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of functional properties .

The STYX solution is typically provided at a concentration of 0.5mg/ml in a buffer containing 20mM Tris-HCl (pH 8.0), 0.15M NaCl, 1mM DTT, and 40% glycerol . This formulation helps maintain protein stability during storage and handling. When working with the protein, it should be thawed gently on ice and diluted in appropriate buffers immediately before use to minimize degradation .

How can STYX Human (26-223) be used in substrate-trapping experiments?

STYX Human (26-223) offers unique advantages in substrate-trapping experiments due to its ability to bind phosphorylated substrates without catalyzing their dephosphorylation. To effectively use STYX in such experiments, researchers should follow this methodological approach:

• Experimental Setup:

  • Immobilize purified His-tagged STYX (26-223) on Ni-NTA resin or similar affinity matrices

  • Prepare cell lysates under non-denaturing conditions to preserve protein-protein interactions

  • Include phosphatase inhibitors in all buffers to prevent dephosphorylation by endogenous phosphatases

• Binding Protocol:

  • Incubate immobilized STYX with cell lysates at 4°C for 2-4 hours with gentle rotation

  • Wash thoroughly with buffer containing low concentrations of imidazole (10-20 mM) to remove non-specific binding

  • Elute bound complexes with high imidazole (250-300 mM) buffer

• Analysis of Trapped Substrates:

  • Analyze eluted complexes by SDS-PAGE followed by Western blotting with anti-phosphotyrosine or specific substrate antibodies

  • For unbiased discovery, employ mass spectrometry to identify novel binding partners

This approach exploits STYX's unique property as a pseudophosphatase that can form stable complexes with phosphorylated substrates without catalyzing their dephosphorylation, effectively "trapping" them for identification and characterization .

What are the methodological considerations when using STYX (26-223) in phosphatase activity assays?

When using STYX (26-223) in phosphatase activity assays, several methodological considerations must be addressed to obtain reliable and interpretable results:

• Negative Control Design:

  • STYX (26-223) serves as an excellent negative control in phosphatase assays due to its lack of catalytic activity

  • Include equal molar concentrations of STYX and active phosphatases to ensure comparable substrate binding

• Competitive Inhibition Studies:

  • To assess STYX's role as a natural regulator, design competition assays where:

    • Pre-incubate substrate with STYX before adding active phosphatases

    • Monitor reaction rates with varying STYX:phosphatase ratios

    • Plot inhibition curves to determine IC₅₀ values

• Buffer Optimization:

  • Ensure buffer compatibility with both STYX stability and phosphatase activity

  • Standardized buffer composition:

    • 50 mM HEPES (pH 7.2)

    • 150 mM NaCl

    • 5 mM DTT

    • 0.01% Tween-20

• Data Interpretation:

  • Account for the presence of His-tag when interpreting binding affinity data

  • Normalize enzyme activities to protein concentration determined by Bradford or BCA assay

  • Consider that STYX might exhibit substrate specificity despite lacking catalytic activity

By carefully addressing these methodological considerations, researchers can effectively utilize STYX (26-223) to investigate phosphorylation-dependent signaling pathways and regulatory mechanisms .

How can differential scanning fluorimetry be used to analyze STYX (26-223) interactions with potential binding partners?

Differential Scanning Fluorimetry (DSF) provides a powerful approach for analyzing STYX (26-223) interactions with potential binding partners by measuring thermal stability shifts upon ligand binding. The methodology involves:

• Experimental Setup:

  • Prepare STYX (26-223) at 0.1-0.2 mg/ml in buffer without glycerol

  • Add SYPRO Orange dye (final concentration 5-10X)

  • Test potential binding partners at concentrations ranging from 10 μM to 1 mM

  • Include appropriate buffer-only and protein-only controls

• Temperature Gradient Protocol:

  • Start at 25°C and increase to 95°C at a rate of 1°C per minute

  • Record fluorescence at each temperature point using a real-time PCR instrument

  • Track the SYPRO Orange signal (excitation ~470 nm, emission ~570 nm)

• Data Analysis:

  • Calculate the melting temperature (Tm) by determining the inflection point of the melting curve

  • Quantify the ΔTm between STYX alone and STYX with binding partners

  • A positive ΔTm (increased stability) indicates binding interaction

• Binding Affinity Determination:

  • Perform concentration-dependent thermal shifts to generate binding curves

  • Calculate apparent Kd values from the concentration dependence of ΔTm

  • Compare binding affinities across different potential substrates

This method is particularly valuable for STYX research because it allows for:

• High-throughput screening of multiple potential binding partners

• Analysis of interactions under near-physiological conditions

• Detection of both strong and weak binding interactions without requiring substrate labeling

What is the biological significance of STYX's pseudophosphatase activity in cellular signaling?

The biological significance of STYX's pseudophosphatase activity in cellular signaling represents a fascinating area of research with important implications for understanding phosphorylation-dependent regulatory mechanisms:

• Substrate Protection Mechanism:
  STYX can bind to phosphorylated substrates and physically shield them from active phosphatases, thereby extending the half-life of the phosphorylated state . This "phosphorylation protection" mechanism allows for temporal regulation of signaling duration without requiring additional kinase activity.

• Signaling Complex Assembly:
  Despite lacking catalytic activity, STYX can function as a scaffold protein that facilitates the assembly of signaling complexes around phosphorylated substrates . By recruiting additional regulatory proteins to specific phosphorylated residues, STYX may coordinate multiprotein complex formation essential for signal transduction.

• Competitive Regulation:
  STYX can compete with active phosphatases for binding to common substrates, thereby functioning as an endogenous regulator of phosphatase activity . This competitive mechanism provides cells with an additional layer of control over phosphorylation-dependent signaling pathways.

• Evolutionary Adaptation:
  The presence of a glycine instead of the catalytic cysteine residue represents an evolutionary adaptation that converted an ancestral active phosphatase into a regulatory pseudophosphatase . This adaptation allows for specialized regulatory functions distinct from simple catalytic dephosphorylation.

Research has particularly highlighted STYX's role in spermiogenesis, suggesting that its pseudophosphatase activity may be essential for proper sperm development and maturation through precise regulation of phosphorylation states in key substrates .

How can STYX Human (26-223) be used to investigate regulatory mechanisms in spermiogenesis?

STYX Human (26-223) can be strategically employed to investigate regulatory mechanisms in spermiogenesis through several methodological approaches:

• Protein Interaction Mapping:

  • Perform pull-down assays using His-tagged STYX (26-223) with testicular tissue extracts

  • Identify spermiogenesis-specific binding partners via mass spectrometry analysis

  • Validate interactions using reverse co-immunoprecipitation with endogenous proteins

• Spatiotemporal Expression Analysis:

  • Use purified STYX (26-223) to generate specific antibodies for immunohistochemistry

  • Map STYX expression patterns during different stages of spermatid differentiation

  • Correlate STYX localization with phosphorylation state changes during spermiogenesis

• Functional Studies:

  • Design competitive binding assays between STYX (26-223) and active phosphatases

  • Measure the impact on phosphorylation states of key spermiogenesis regulators

  • Experimental setup for in vitro spermatid cultures:

    Condition	STYX Concentration	Phosphatase Inhibitors	Expected Outcome
    Control	None	None	Baseline phosphorylation
    STYX only	1-10 μM	None	Protected phosphorylation
    Phosphatase + STYX	1-10 μM	None	Competitive regulation
    Positive control	None	1 mM sodium orthovanadate	Maximal phosphorylation

• Substrate Identification:

  • Incubate STYX (26-223) with differentiating spermatid lysates

  • Analyze bound proteins by phosphoproteomic approaches

  • Quantify differential binding during key developmental transitions

These approaches leverage STYX's unique properties as a pseudophosphatase to identify specific phosphorylation-dependent pathways crucial for sperm development and maturation, potentially revealing novel regulatory mechanisms underlying male fertility .

What are the challenges in distinguishing between substrate binding and catalytic activity when studying STYX?

Distinguishing between substrate binding and catalytic activity when studying STYX presents several methodological challenges that researchers must address through careful experimental design:

• Binding vs. Activity Differentiation:

  • STYX can bind phosphorylated substrates without catalyzing dephosphorylation, requiring assays that separate these functions

  • Implement endpoint phosphatase assays with extended incubation times (up to 24 hours) to detect even minimal catalytic activity

  • Use non-hydrolyzable phosphate analogs to differentiate binding from potential residual catalytic activity

• Kinetic Analysis Limitations:

  • Standard Michaelis-Menten kinetics are not applicable due to STYX's lack of catalytic turnover

  • Replace with equilibrium binding assays such as:

    • Isothermal titration calorimetry (ITC)

    • Surface plasmon resonance (SPR)

    • Microscale thermophoresis (MST)

• Structural Considerations:

  • The binding pocket of STYX may exhibit different conformational properties compared to active phosphatases

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational dynamics upon substrate binding

  • Compare binding pocket accessibility between STYX and active phosphatases using molecular probes

• Competition Assay Design:
  When using competition assays to indirectly measure STYX binding:

  Parameter	Critical Consideration	Methodological Solution
  Stoichiometry	Equal molar ratios between STYX and active phosphatase	Precise protein quantification
  Binding kinetics	Different on/off rates may confound interpretation	Time-course measurements
  Substrate concentration	Must be below Km for accurate competition	Pilot titration experiments
  Buffer conditions	May differentially affect STYX and active phosphatases	Standardized buffer optimization

• His-tag Interference:

  • The N-terminal His-tag may influence binding properties

  • Compare results between His-tagged and tag-cleaved versions of STYX (26-223)

  • Control for non-specific interactions due to the His-tag using irrelevant His-tagged proteins

Addressing these challenges requires integrating multiple complementary approaches to build a comprehensive understanding of STYX's substrate binding properties distinct from catalytic activity .

How does the His-tag affect the binding properties of STYX (26-223) in protein interaction studies?

The N-terminal His-tag present in recombinant STYX (26-223) can significantly impact protein interaction studies in several ways that researchers should consider when designing experiments and interpreting results:

• Electrostatic Interference:

  • The His-tag introduces six consecutive positively charged histidine residues that can alter the protein's surface charge distribution

  • This may create non-physiological electrostatic interactions, particularly with negatively charged substrates

  • Recommendation: Compare binding affinities with and without His-tag cleavage using a controlled panel of substrates

• Steric Hindrance Effects:

  • The 23 amino acid N-terminal extension (MGSSHHHHHH SSGLVPRGS) can potentially:

    • Block access to binding sites near the N-terminus

    • Alter protein conformation through additional flexibility

    • Create artificial binding surfaces not present in native STYX

  • Methodology: Employ hydrogen-deuterium exchange mass spectrometry to map conformational changes induced by the tag

• Metal Ion Coordination:

  • Histidine residues have high affinity for divalent metal ions

  • This can lead to:

    • Artificial protein-protein cross-linking via metal bridges

    • Altered binding to metalloproteins

    • Interference with assays involving metal-dependent processes

• Experimental Controls:
  To mitigate His-tag effects, implement the following controls:

  Control Type	Implementation	Purpose
  Tag-cleaved control	Use precision proteases (TEV/HRV 3C) to remove tag	Direct comparison with tagged protein
  Competitive elution	Include imidazole gradient in binding studies	Distinguish tag-dependent from tag-independent interactions
  Alternative tag position	Compare N-terminal vs. C-terminal His-tag	Identify position-specific effects
  Alternative tag chemistry	Compare His-tag with other affinity tags (GST, MBP)	Distinguish tag-specific artifacts

• Quantitative Compensation:

  • When tag removal is not feasible, mathematically correct for His-tag effects by:

    • Determining the binding contribution of the tag alone using control peptides

    • Implementing correction factors in binding affinity calculations

    • Using computational modeling to predict and account for tag influences

These considerations are particularly important for STYX (26-223) research, as its primary function involves protein-protein interactions that could be subtly altered by the presence of the His-tag .

What are the latest methodological approaches for using STYX (26-223) in phosphoproteomic studies?

Recent advances have expanded the methodological toolkit for employing STYX (26-223) in phosphoproteomic studies, offering researchers sophisticated approaches to explore phosphorylation-dependent cellular processes:

• STYX-based Substrate Identification:

  • Immobilize His-tagged STYX (26-223) on functionalized agarose beads

  • Incubate with cellular lysates under native conditions

  • Perform sequential elutions with:

    • Low stringency buffer (150 mM NaCl) for weak interactions

    • Medium stringency buffer (300 mM NaCl) for moderate interactions

    • High stringency buffer (500 mM NaCl with 250 mM imidazole) for strong interactions

  • Analyze eluted fractions by mass spectrometry with titanium dioxide enrichment for phosphopeptides

• Proximity-dependent Labeling:

  • Generate STYX-BioID or STYX-TurboID fusion constructs

  • Express in relevant cell types (particularly testicular cell lines for spermiogenesis studies)

  • Induce proximity-dependent biotinylation of proteins interacting with STYX

  • Purify biotinylated proteins and perform phosphoproteomic analysis

  • Compare interactome under various cellular conditions

• Phospho-competitive Binding Assays:

  • Design synthetic phosphopeptide libraries representing potential STYX substrates

  • Implement bead-based multiplexed binding assays with fluorescent readouts

  • Quantify binding affinity (Kd values) for each phosphopeptide

  • Generate phospho-binding motif preferences for STYX using position-specific scoring matrices

• Integrated Phosphoproteomics Workflow:

  Stage	Methodology	Analytical Output
  Enrichment	STYX affinity chromatography	STYX-binding phosphoproteome
  Identification	LC-MS/MS with IMAC enrichment	Phosphosite mapping
  Validation	Synthesized phosphopeptide arrays	Binding specificity profiles
  Functional analysis	STYX overexpression/knockdown	Phosphorylation dynamics
  Network integration	Computational pathway analysis	Regulatory node identification

• CRISPR-based Functional Proteomics:

  • Generate STYX knockout cell lines using CRISPR/Cas9

  • Perform quantitative phosphoproteomics comparing wild-type and knockout cells

  • Rescue experiments with recombinant STYX (26-223)

  • Identify phosphosites with altered occupancy dependent on STYX expression

These advanced methodological approaches enable researchers to move beyond simple binding studies toward comprehensive understanding of STYX's role in regulating phosphorylation-dependent cellular processes, particularly in specialized contexts like spermiogenesis .

How can structural studies of STYX (26-223) inform the development of phosphatase inhibitors?

Structural studies of STYX (26-223) provide unique insights that can inform rational design of phosphatase inhibitors through several methodological approaches:

• Comparative Structural Analysis:

  • Determine the crystal structure of STYX (26-223) bound to phosphorylated substrates

  • Compare binding pocket architecture with active phosphatases

  • Identify key structural differences that enable substrate binding without catalysis

  • Map the molecular features that could be mimicked in synthetic inhibitor design

• Binding Pocket Characterization:

  • Perform site-directed mutagenesis of residues surrounding the glycine that replaces the catalytic cysteine

  • Measure effects on binding affinity using isothermal titration calorimetry

  • Determine the contribution of individual residues to substrate recognition

  • Generate a detailed pharmacophore model of the binding site

• Fragment-based Drug Design Approach:

  • Use STYX (26-223) as a molecular template for rational inhibitor design

  • Screen fragment libraries against STYX binding pocket

  • Identify molecular scaffolds that mimic STYX's binding mode

  • Optimize fragments for enhanced interaction with active phosphatases

• Structure-Activity Relationship Studies:

  Structural Element	Role in STYX	Implication for Inhibitor Design
  Glycine substitution	Eliminates catalysis	Create non-hydrolyzable phosphate mimetics
  Binding pocket topology	Preserves substrate recognition	Design complementary inhibitor scaffolds
  N-terminal region	Contributes to binding specificity	Include mimetic elements in inhibitor structure
  Surface loops	Determine substrate access	Target specific phosphatase loop configurations

• Molecular Dynamics Simulations:

  • Perform comparative molecular dynamics simulations of:

    • STYX (26-223) bound to substrates

    • Active phosphatases with the same substrates

    • Active phosphatases with potential inhibitors

  • Analyze binding energy landscapes and conformational changes

  • Identify transient binding states that could be exploited for inhibitor design

  • Predict inhibitor specificity across phosphatase families

The structural insights derived from STYX (26-223) are particularly valuable because they reveal how nature has evolved a protein that binds phosphorylated substrates without catalyzing their dephosphorylation—precisely the property sought in effective phosphatase inhibitors .

What are common challenges in purifying active STYX (26-223) and how can they be addressed?

Obtaining high-quality, active STYX (26-223) recombinant protein presents several technical challenges that researchers should anticipate and address through optimized purification protocols:

• Protein Solubility Issues:

  • Challenge: STYX (26-223) may form inclusion bodies during E. coli expression

  • Solution:

    • Lower induction temperature to 16-18°C

    • Reduce IPTG concentration to 0.1-0.2 mM

    • Co-express with chaperones (GroEL/ES, DnaK/J)

    • Consider fusion partners (MBP, SUMO) to enhance solubility

• Protein Stability During Purification:

  • Challenge: STYX may be susceptible to aggregation during purification steps

  • Optimization approach:

    Buffer Component	Recommended Range	Rationale
    NaCl	150-300 mM	Prevents non-specific interactions
    Glycerol	10-40%	Stabilizes protein structure
    DTT	1-5 mM	Prevents oxidation of cysteine residues
    EDTA	0.5-1 mM	Chelates metal ions that may promote aggregation
    pH	7.5-8.0	Maintains optimal protein folding

• His-tag Accessibility Challenges:

  • Challenge: N-terminal His-tag may become occluded during protein folding

  • Solutions:

    • Include denaturing agents (1-2 M urea) during initial binding

    • Try extended binding times (4-16 hours) at 4°C

    • Consider C-terminal His-tag constructs as alternatives

    • Implement on-column refolding protocols if necessary

• Contaminant Removal:

  • Challenge: Co-purifying E. coli proteins may bind to affinity matrices

  • Optimization strategies:

    • Implement two-step purification (IMAC followed by size exclusion)

    • Include imidazole wash steps (20-40 mM) to remove weakly bound contaminants

    • Add high salt washes (0.5-1 M NaCl) to disrupt ionic interactions

    • Consider ion exchange chromatography as a polishing step

• Quality Control Metrics:

  • Verify proper folding using circular dichroism spectroscopy

  • Confirm binding activity with model phosphopeptides

  • Assess monodispersity by dynamic light scattering

  • Validate identity by mass spectrometry and N-terminal sequencing

By implementing these optimization strategies, researchers can overcome common purification challenges to obtain high-quality STYX (26-223) recombinant protein suitable for downstream functional and structural studies .

How can researchers validate that STYX (26-223) is properly folded and functionally active?

Validating the proper folding and functional activity of purified STYX (26-223) recombinant protein is critical for reliable experimental outcomes. A comprehensive validation approach should include:

• Structural Integrity Assessment:

  • Circular Dichroism (CD) Spectroscopy:

    • Measure far-UV (190-250 nm) spectrum to assess secondary structure

    • Compare with predicted structural elements based on homology models

    • Monitor thermal stability by temperature-dependent CD measurements

  • Fluorescence Spectroscopy:

    • Measure intrinsic tryptophan fluorescence (excitation 280 nm, emission 300-400 nm)

    • Assess tertiary structure integrity through spectral characteristics

    • Compare with denatured protein as negative control

• Homogeneity and Aggregation Analysis:

  • Size Exclusion Chromatography (SEC):

    • Verify monodisperse elution profile corresponding to expected molecular weight

    • Quantify percentage of aggregates or degradation products

  • Dynamic Light Scattering (DLS):

    • Determine hydrodynamic radius and polydispersity index

    • Acceptable criteria: polydispersity <15%, radius consistent with monomeric state

• Functional Binding Validation:

  Binding Assay Type	Methodology	Expected Outcome for Active STYX
  Phosphopeptide binding	Fluorescence polarization	Kd values in 1-10 μM range
  Protein substrate binding	Pull-down with immobilized STYX	Enrichment of known partners
  Competition assay	Displacement of labeled probe	IC50 values consistent with literature
  Surface Plasmon Resonance	Immobilized STYX with substrate flow	Association/dissociation kinetics

• Thermal Stability Assessment:

  • Differential Scanning Fluorimetry (DSF):

    • Determine melting temperature (Tm) using SYPRO Orange dye

    • Properly folded STYX should have Tm >45°C

    • Verify thermal shift in presence of binding partners

    • Expected ΔTm of 2-5°C upon substrate binding indicates functional binding pocket

• Comparative Benchmark Analysis:

  • Compare binding properties with commercially validated references

  • Verify consistent performance across different production batches

  • Establish internal quality control standards for:

    • Minimum purity (>90% by SDS-PAGE)

    • Binding affinity thresholds (within 2-fold of reference values)

    • Thermal stability parameters (Tm within ±2°C of reference)

These validation approaches provide complementary information about both structural integrity and functional activity, ensuring that STYX (26-223) is suitable for reliable downstream experimental applications .