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| Product Name | Glutathione Magnetic Beads (GST-tag Purification) |
| Catalog No. | MAGBEA-0015 |
| Description | Glutathione-functionalized superparamagnetic beads for the affinity purification of GST-tagged recombinant proteins. Reduced glutathione is covalently linked to the bead surface through a stable spacer arm via epoxy or NHS coupling chemistry. The immobilized glutathione provides high-specificity binding to glutathione S-transferase (GST, ~26 kDa) fusion proteins, enabling one-step purification from crude cell lysates with gentle, non-denaturing reduced glutathione elution that preserves protein function. |
| Intended Use | Purification of GST-fusion proteins from bacterial, yeast, and mammalian expression systems for biochemical characterization, protein-protein interaction studies (GST pull-down assays), antibody production, and enzymatic research. |
| Principle / Technology | Reduced glutathione (γ-Glu-Cys-Gly tripeptide) is covalently immobilized on the bead surface through its amino terminus via a 6-10 carbon spacer arm. The GST enzyme (~26 kDa from Schistosoma japonicum) binds glutathione with high specificity (Kd ~10^-5 M) through its active site. After washing away contaminating proteins, the GST-fusion protein is eluted by competitive displacement with 10-50 mM reduced glutathione under non-denaturing conditions at neutral to mildly alkaline pH, preserving the native structure and enzymatic activity of the fusion partner. |
| Detection Method | BCA and Bradford assays for protein quantitation; SDS-PAGE with Coomassie staining for purity; Western blot with anti-GST antibody; GST enzymatic activity assay (CDNB substrate); glutathione loading verification by TNBS or Ellman's reagent; DLS for bead size |
| Sample Type | GST-fusion proteins expressed in E. coli, yeast (S. cerevisiae, P. pastoris), insect cells, and mammalian cells; cell lysates, periplasmic fractions, and secreted proteins; compatible with GST tag at N-terminus or C-terminus of fusion partner |
| Performance Range / Specifications | Bead diameter: 1-3 µm; glutathione density: 5-15 µmol/mL settled beads; GST binding capacity: 5-15 mg GST per mL settled beads; binding constant (Kd): ~10^-4 to 10^-5 M; elution efficiency: >90% with 10-50 mM reduced glutathione at pH 8.0; non-specific binding: <5%; magnetic separation: <15 seconds |
| Sensitivity / LOD | GST protein detection from lysates with <5 µg/mL target; pull-down sufficient for MS identification of protein interaction partners; GST enzymatic activity (CDNB assay) linear with bead-bound protein amount |
| Specificity | Specific binding to GST and GST-fusion proteins; no binding of non-GST endogenous bacterial proteins; GST does not bind oxidized glutathione (GSSG)—use only reduced form for elution; spacer arm minimizes steric hindrance for large fusion partners; no cross-reactivity with other affinity tags (His6, MBP, FLAG) |
| Reaction Conditions / Protocol | Equilibrate beads with PBS pH 7.4; add clarified cell lysate; incubate 30-60 minutes at room temperature or 1-2 hours at 4°C with rotation; magnetically separate; wash 3-5 times with PBS; elute with 10-50 mM reduced glutathione in 50 mM Tris-HCl pH 8.0; incubate 10-15 minutes; repeat elution for maximum recovery; total protocol: 1-2 hours; beads regenerable 3-5 times |
| Components / Formulation | Glutathione-functionalized superparamagnetic beads, 25-50% slurry in 20% ethanol or PBS with 0.02% sodium azide; reduced glutathione (for elution) not included; PBS and Tris buffers not included |
| Storage Conditions | 2-8°C in 20% ethanol or PBS/azide; do not freeze; protect from light; for long-term storage, 20% ethanol preferred; glutathione is susceptible to oxidation—store under conditions that minimize oxidation |
| Shelf Life | 18 months at 2-8°C in 20% ethanol; 12 months in PBS/azide; glutathione oxidation monitored; binding capacity >85% at 18 months; glutathione leaching <5%; preservative effective through expiry |
| Package Specifications | 1 mL, 5 mL, 10 mL, 25 mL settled bead volume; bulk and OEM packaging; trial pack for method development; pre-packed column format also available |
| Product Form | White to off-white bead suspension in storage buffer; homogeneous after gentle shaking; clear supernatant after magnetic separation; no discoloration (yellowing indicates glutathione oxidation); uniform spherical particles |
| Quality Control | Per lot: glutathione density, GST binding capacity (recombinant GST standard), elution efficiency with reduced glutathione, non-specific binding, glutathione leaching, GST enzymatic activity of purified protein, endotoxin, sterility, particle size |
| Key Features | Reduced glutathione for specific, gentle GST elution; covalent glutathione linkage for low leaching; spacer arm for efficient large fusion protein binding; mild elution preserves protein activity; validated for GST pull-down assays and protein interaction studies; regenerable for cost-effective repeated use |
| Purity | Glutathione >90% coupling efficiency; endotoxin <0.1 EU/mg; no oxidized glutathione by HPLC; GST purity >90% by SDS-PAGE from single-step purification |
| Concentration | 25-50% slurry; glutathione 5-15 µmol/mL settled beads; GST binding 5-15 mg/mL; exact values per lot |
| Activity / Unit Definition | GST binding: 5-15 mg/mL settled beads; elution >90% with 50 mM reduced glutathione; regenerable 3-5 times with >80% capacity retention; GST enzymatic activity (CDNB) maintained in eluate |
| Molecular Weight | Glutathione: ~307 Da (reduced form); GST: ~26 kDa monomer (~52 kDa dimer); spacer arm: ~150-200 Da |
| Source / Origin | Synthetic reduced glutathione (pharmaceutical grade); chemical coupling via epoxy or NHS-activated linker to polymer-magnetite beads; all components synthetic; no animal-derived materials; recombinant GST used for QC testing expressed in E. coli |
| pH Range / Optimal pH | Binding: pH 6.5-8.5 (optimal 7.0-7.5); elution: pH 7.5-8.5 (optimal 8.0); glutathione oxidation increases below pH 5 and above pH 9; bead integrity: pH 2-13; avoid prolonged exposure to pH <4 |
| Shipping Conditions | Ambient temperature in 20% ethanol; cold packs optional; non-hazardous; standard courier; protect from extreme heat and freezing |
| Expiration Date / Stability | 18 months in 20% ethanol at 2-8°C; glutathione stability monitored by DTNB assay; binding capacity >85% at expiry; oxidation prevented by ethanol storage; opened product: 6 months with proper handling |
| Regulatory / Compliance | For research use only; not for GMP or therapeutic production; ISO 9001 manufacturing; certificate of analysis per lot; material safety data sheet available |
| Compatibility | Compatible with PBS, Tris, HEPES lysis buffers; tolerant to 150-500 mM NaCl; 0.1-1% Triton X-100, NP-40, Tween-20; DTT up to 5 mM (reduces glutathione—may cause leaching at high concentration); EDTA up to 5 mM; avoid strong oxidizing agents; compatible with protease inhibitor cocktails |
| Recommended Buffer System | Binding/wash: PBS pH 7.4 or 50 mM Tris-HCl pH 8.0, 150 mM NaCl; elution: 50 mM Tris-HCl pH 8.0, 10-50 mM reduced glutathione (prepare fresh); storage: 20% ethanol or PBS/azide; reduced glutathione solution should be prepared immediately before use and pH adjusted to 8.0 |
| Application Notes / Precautions | Prepare reduced glutathione elution buffer fresh daily (oxidation reduces elution efficiency); for sensitive proteins, reduce glutathione concentration to 10 mM; dialyze or desalt eluate to remove glutathione if it interferes with downstream assays; avoid Tris buffers above pH 8.5 during binding; for GST pull-down, block beads with BSA before incubation to reduce non-specific binding; glutathione beads can be stripped with 6 M guanidine HCl for deep cleaning between uses |
| Batch-to-Batch Consistency | Glutathione density CV <15%; GST binding CV <12%; elution efficiency >90% all lots; glutathione leaching <5% per lot; each lot validated with recombinant GST standard |
For research use only, not for clinical use.
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