S100B Human, His

What are the key binding partners and functional roles of S100B in neural tissues?

S100B demonstrates multiple functional roles through its interactions with various protein targets. It binds to and initiates the activation of STK38 by releasing autoinhibitory intramolecular interactions within the kinase . The protein interacts with RAGE receptors, potentially mediating calcium-dependent regulation of physiological processes by modulating the activity of TPR-containing proteins . In neural tissues, S100B acts as a neurotrophic factor promoting astrocytosis and axonal proliferation. Additional functions include:

• Facilitating ATAD3A cytoplasmic processing and mitochondrial localization

• Promoting sympathetic innervation of adipose tissue

• Involvement in myocyte apoptosis through ERK1/2 and p53/TP53 signaling after myocardial infarction

• Regulating neurite extension

• Inhibiting PKC-mediated phosphorylation and microtubule assembly

What expression systems are optimal for producing high-quality recombinant human S100B with His-tag?

Escherichia coli represents the most commonly utilized expression system for recombinant human S100B with His-tag due to its ability to produce high yields of soluble protein with ≥95% purity . When designing expression constructs, researchers should consider:

• Codon optimization for E. coli expression

• Proper positioning of the His-tag (N- or C-terminal) to minimize functional interference

• Inclusion of a protease cleavage site if tag removal is required

• Temperature optimization during induction (typically 25-30°C) to maximize soluble protein yield

• IPTG concentration optimization for induction

For applications requiring post-translational modifications, mammalian expression systems may be considered, though yields are typically lower than bacterial systems.

What purification protocols yield the highest purity and activity of His-tagged S100B?

A multi-step purification protocol is recommended to achieve ≥95% purity while maintaining protein activity:

• Initial clarification: Centrifugation of bacterial lysate at 12,000-15,000g for 30 minutes to remove cellular debris

• Immobilized metal affinity chromatography (IMAC): Using Ni-NTA resin with optimized imidazole gradients (typically 10-20mM for binding, 250-300mM for elution)

• Size exclusion chromatography: To separate dimeric S100B from aggregates and other impurities

• Quality control: SDS-PAGE and Western blot analysis to confirm purity and identity

• Activity assessment: Calcium-binding assays or functional interaction studies

Buffer optimization is critical, typically including 20-50mM Tris-HCl (pH 7.5-8.0), 100-300mM NaCl, and potentially low concentrations of reducing agents like DTT or β-mercaptoethanol to prevent oxidation of cysteine residues.

How can researchers effectively address protein aggregation issues during S100B purification?

S100B aggregation during purification can significantly impact downstream applications. Methodological approaches to minimize aggregation include:

• Maintaining appropriate buffer conditions (pH 7.5-8.0) during all purification steps

• Including 5-10% glycerol in purification buffers to enhance protein stability

• Controlling calcium levels, as excessive calcium can promote aggregation (consider using EGTA during initial purification steps)

• Maintaining lower protein concentrations during purification steps

• Performing purification at 4°C whenever possible

• Considering the addition of non-ionic detergents (0.01-0.05% Tween-20) for highly concentrated preparations

• Implementing filtration steps (0.22μm) prior to final storage

• Lyophilization of purified protein in the presence of stabilizing agents for long-term storage

What analytical techniques are most effective for characterizing recombinant S100B-His protein quality?

Multiple complementary analytical techniques should be employed for comprehensive quality assessment:

• SDS-PAGE and Western blotting: To confirm molecular weight, purity, and immunoreactivity using anti-S100B and anti-His antibodies

• Mass spectrometry: For accurate molecular weight determination and confirmation of sequence integrity

• Dynamic light scattering (DLS): To assess protein homogeneity and detect potential aggregation

• Circular dichroism (CD) spectroscopy: To verify proper secondary structure formation

• Fluorescence spectroscopy: To evaluate tertiary structure and monitor calcium/zinc binding through intrinsic tryptophan fluorescence

• Functional binding assays: Such as ELISA-based binding to RAGE protein to confirm biological activity

• Thermal shift assays: To assess protein stability under various buffer conditions

Researchers should establish acceptance criteria for each analytical parameter based on their specific experimental requirements.

How do storage conditions affect S100B-His stability and activity?

Storage conditions significantly impact S100B-His stability and activity. Based on research practices, optimal storage approaches include:

• Short-term storage (1-2 weeks): 4°C in buffer containing 20-50mM Tris-HCl pH 7.5-8.0, 100-150mM NaCl with addition of protease inhibitors

• Medium-term storage (1-6 months): -20°C or -80°C in storage buffer supplemented with 10-20% glycerol

• Long-term storage (>6 months): Lyophilized form at -80°C after addition of stabilizers like trehalose or sucrose

Researchers should avoid:

• Multiple freeze-thaw cycles (aliquot before freezing)

• Prolonged exposure to room temperature

• Extreme pH conditions (<pH 6.0 or >pH 8.5)

• High concentrations of reducing agents that may disrupt structural disulfide bonds

Activity testing after storage is recommended through functional assays such as calcium-binding assessment or RAGE interaction studies.

How can researchers effectively use S100B-His in protein-protein interaction studies?

S100B-His serves as an excellent tool for protein-protein interaction studies due to its well-characterized binding partners. Methodological approaches include:

• Pull-down assays: Utilizing the His-tag to immobilize S100B on Ni-NTA or cobalt resins, followed by incubation with target protein candidates and subsequent identification by mass spectrometry

• Surface Plasmon Resonance (SPR): For real-time binding kinetics assessment and quantitative affinity determination between S100B and binding partners

• Microscale Thermophoresis (MST): For measuring interactions in solution with minimal protein consumption

• Isothermal Titration Calorimetry (ITC): To determine complete thermodynamic profiles of S100B interactions

• Proximity-based assays: Such as FRET or BRET when working with fluorescently labeled interaction partners

When conducting these studies, researchers should consider the calcium and zinc dependency of many S100B interactions, maintaining appropriate divalent cation concentrations in experimental buffers (typically 1-2mM calcium for calcium-dependent interactions) .

What are the key considerations when using S100B-His in structural biology studies?

For structural biology applications with S100B-His, researchers should consider:

• Crystallography preparations:

  • Higher protein purity (>98%) is typically required

  • Tag removal may be necessary using specific proteases

  • Screening of various crystallization conditions including different calcium and zinc concentrations

  • Co-crystallization with binding partners to capture interaction interfaces

• NMR studies:

  • Isotopic labeling (15N, 13C) strategies when expressing in E. coli

  • Optimization of protein concentration and buffer conditions to minimize aggregation

  • Consideration of the dimeric nature of S100B in data interpretation

• Cryo-EM applications:

  • Sample homogeneity is critical

  • Higher molecular weight complexes with binding partners may be preferred

  • Grid preparation optimization to overcome size limitations

• Tag positioning strategy:

  • C-terminal His-tags typically interfere less with S100B function

  • Consider TEV or other protease cleavage sites for tag removal when necessary

What are the methodological considerations for using S100B as a biomarker in neurological disorders?

S100B has been extensively studied as a biomarker in various neurological conditions including Alzheimer's disease, Parkinson's disease, and ALS . Key methodological considerations include:

• Sample type selection:

  • Cerebrospinal fluid (CSF) offers higher sensitivity but requires invasive collection

  • Serum/plasma provides easier collection but may have lower specificity

  • Consideration of other biological fluids (saliva, urine) based on research question

• Pre-analytical variables:

  • Standardized collection protocols to minimize external S100B release

  • Consistent sample processing timeframes

  • Appropriate storage temperatures (-80°C preferred for long-term)

  • Consideration of freeze-thaw effects on S100B stability

• Analytical methods:

  • ELISA remains the gold standard for quantification

  • Electrochemiluminescence immunoassays for enhanced sensitivity

  • Mass spectrometry for absolute quantification in complex matrices

  • Point-of-care testing systems for rapid assessment

• Reference range establishment:

  • Age and sex-specific reference ranges

  • Disease-specific cutoff values

  • Consideration of comorbidities affecting baseline levels

• Longitudinal monitoring strategies:

  • Optimized sampling intervals

  • Integration with other biomarkers for improved specificity

How can researchers optimize S100B detection protocols in sports-related concussion research?

Sports-related concussion research requires specialized approaches to S100B measurement:

• Timing of sample collection is critical:

  • Baseline measurements during pre-season

  • Acute post-injury sampling within 3-6 hours

  • Follow-up sampling at standardized intervals (24h, 72h, 7d)

• Consideration of confounding factors:

  • Physical activity itself increases S100B (0.1 μg/L cutoff may not be suitable)

  • Different sports produce different baseline elevations

  • Individual athlete variability requires personalized baselines

  • Body contact without head impact may elevate S100B

• Methodology standardization:

  • Consistent analytical platforms across studies

  • Standardized sample processing protocols

  • Uniform reporting units (μg/L preferred)

  • Integration with clinical assessment tools

• Research design recommendations:

  • Matched controls based on sport type, position, age, sex, and race

  • Longitudinal monitoring within individuals

  • Multimodal assessment including other biomarkers and clinical measures

The current literature indicates that while the 0.1 μg/L cutoff for S100B has excellent negative predictive value for concussion, its positive predictive value requires improvement through integration with other clinical data and biomarkers .

How can S100B-His be utilized in developing targeted therapeutics for neurological disorders?

S100B represents a promising target for therapeutic development in neurological disorders given its involvement in pathological processes. Advanced research approaches include:

• Small molecule inhibitor development:

  • Structure-based design targeting calcium-binding domains

  • In vitro screening assays using recombinant S100B-His

  • Assessing compound effects on S100B-RAGE interactions

  • Testing inhibitors in cellular models of neurodegeneration

• Antibody-based interventions:

  • Development of blocking antibodies against S100B

  • Epitope mapping using recombinant S100B-His variants

  • Functional neutralization assays

  • Assessment of blood-brain barrier penetration

• S100B-mediated drug delivery systems:

  • Coupling of therapeutic agents to S100B-binding peptides

  • Targeting astrocyte-specific delivery

  • Assessment of cellular uptake and distribution

  • Evaluation of therapeutic efficacy in disease models

• Peptide-based therapies:

  • Design of peptides mimicking S100B interaction domains

  • Competitive binding assays with recombinant S100B-His

  • Optimization of peptide stability and delivery

  • In vivo testing in animal models of neurological disease

What advanced experimental designs can resolve contradictory findings in S100B research?

The S100B literature contains some contradictory findings regarding its roles in neuroprotection versus neurotoxicity. Advanced experimental approaches to resolve these contradictions include:

• Concentration-dependent studies:

  • Precise dose-response experiments with nanomolar versus micromolar S100B concentrations

  • Monitoring different signaling pathways activated at different concentrations

  • Time-course analyses to distinguish acute versus chronic effects

• Cell-type specific investigations:

  • Comparison of S100B effects across different neural cell populations

  • Co-culture systems to assess cell-cell communication

  • Conditional expression systems for temporal control

• Advanced animal models:

  • Conditional and inducible S100B knockout/knockin models

  • Cell-type specific S100B modulation in vivo

  • Integration of multiple physiological readouts

  • Humanized animal models for improved translation

• Multi-omics approaches:

  • Integration of transcriptomics, proteomics, and metabolomics

  • Network analysis to identify context-dependent signaling nodes

  • Single-cell analyses to resolve population heterogeneity

  • Temporal profiling to identify critical transition points in disease progression

These advanced experimental designs can help clarify the dual roles of S100B in neuroprotection and neurotoxicity, which appear to be context-dependent and influenced by factors such as concentration, duration of exposure, and the specific cellular microenvironment.

How can researchers address inconsistent S100B-His activity in experimental systems?

Inconsistent activity of recombinant S100B-His in experimental systems can significantly impact research outcomes. Methodological approaches to troubleshoot include:

• Protein quality assessment:

  • Verification of proper folding through circular dichroism

  • Assessment of dimer formation via native PAGE or size exclusion chromatography

  • Confirmation of calcium binding capacity through functional assays

  • Evaluation of batch-to-batch consistency

• Buffer optimization strategies:

  • Systematic testing of different buffer compositions

  • Adjustment of calcium and zinc concentrations for optimal activity

  • Consideration of physiologically relevant conditions

  • Addition of stabilizing agents if necessary

• Storage and handling improvements:

  • Implementation of single-use aliquots to avoid freeze-thaw cycles

  • Validation of activity retention after different storage periods

  • Temperature logging during sample handling

  • Standardization of reconstitution protocols for lyophilized protein

• Experimental design refinements:

  • Inclusion of appropriate positive and negative controls

  • Dose-response characterization for each experimental system

  • Time-course studies to determine optimal treatment duration

  • Validation across multiple detection methods

What strategies can optimize detection sensitivity in S100B biomarker research?

Enhancing detection sensitivity is critical for S100B biomarker applications, particularly when measuring low concentrations in biological samples. Optimization strategies include:

• Sample preparation enhancements:

  • Pre-concentration techniques for dilute samples

  • Removal of interfering proteins through immunodepletion

  • Optimization of extraction protocols for different biological matrices

  • Standardization of centrifugation and filtration steps

• Advanced detection methodologies:

  • Implementation of ultrasensitive ELISA formats (chemiluminescent, fluorescent)

  • Digital ELISA platforms for single-molecule detection

  • Mass spectrometry with targeted multiple reaction monitoring

  • Signal amplification strategies (e.g., tyramide signal amplification)

• Assay component optimization:

  • Antibody affinity maturation or alternative binder development

  • Blocking optimization to reduce background

  • Incubation condition refinement (time, temperature, agitation)

  • Detection enzyme or fluorophore selection for maximum sensitivity

• Data analysis approaches:

  • Application of advanced curve-fitting algorithms

  • Digital signal processing to enhance signal-to-noise ratio

  • Machine learning algorithms for complex pattern recognition

  • Integration of multiple biomarkers for improved diagnostic accuracy

These strategies can help researchers achieve detection limits in the pg/mL range, which is particularly important for early disease detection and monitoring subtle changes in S100B levels during disease progression or therapeutic intervention.