Glutathione Resin

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Spezifität Glutathione Resin is an affinity chromatography medium designed for easy, one-step purification of recombinant glutathione S-transferase (GST) fusion proteins and other glutathione binding proteins expressed in E. coli, insect cells and mammalian cells. The recombinant GST fusion proteins can be purified directly from pre-treated cell lysate using Glutathione Resin. It is the excellent choice for high performance purifications.
Produktmerkmale Resin volume: 10 ml settled resin (20 ml 50% slurry)
Ligand: Glutathione
Dynamic binding capacity: > 20 mg horse liver GST (26 kDa)/ml settled resin
Matrix: 4% cross-linked agarose
Average particle size: 90 μm

Key features:
Easy to use: Simple and fast procedures for purification of GST-fusion proteins
High capacity: The system can support over 20 mg horse liver GST/ml medium
Stability: This reusable resin shows no obvious decrease of the binding capacity after three uses
Bead Ligand Glutathione
Bead Matrix Agarose beads
Bead Size 90 µm
Aufbereitung der Reagenzien

Regeneration and Storage of Glutathione Resin
Glutathione Resin can be reused to purify the same protein three times without regeneration. If the target GST-fusion protein is different, however, the Glutathione Resin must be regenerated using the following protocol:
1. Wash the column with 2×bed volumes of 0.1 M Tris HCl + 0.5 M NaCl, pH 8.5.
2. Wash the column with 2×bed volumes of 0.1 M sodium acetate + 0.5 M NaCl, pH 4.5.
3. Re-equilibrate the column with 3-5×bed volumes of 1×PBS.
4. For long-term storage, the resin should be stored in 1×PBS containing 20% ethanol at 2 - 8°C.

Aufbereitung der Proben

Preparation of Cell Extract
1.Harvest cells by centrifugation at 3,000 g at 4°C for 10 min, remove and discard the supernatant.
2. Resuspend the cell pellet in 3 ml ice-cold 1×PBS buffer per 50 ml culture and centrifuge at 3,000 g at 4°C for 10 min. Remove and discard the supernatant.
3. Freeze the cell pellet at -80°C for 1 hour (This is also a convenient point to stop and one can continue the procedure later).
4. Thaw cell pellet on ice and resuspend cells in 3 ml of ice-cold 1×PBS buffer per 50 ml culture. If desired, add appropriate additives, such as non-ionic detergents (NP-40) or protease inhibitors (PMSF).
5. Disrupt cells by brief pulses of sonication on ice until the sample is no longer viscous.
6. Centrifuge at 12,000 g at 4°C for 10 min and carefully transfer the supernatant (soluble fraction) to a clean and pre-chilled tube and resuspend pellet (insoluble fraction) with 3 ml of ice-cold 1×PBS buffer per 50 ml culture.
7. Aliquot 10 μl samples from both soluble and insoluble fractions for SDS-PAGE analysis [ by adding equal volume of 2X SDS Sample Buffer (125 mM Tris-HCl, pH 6.8, 4% w/v SDS, 20% glycerol, 100 mM DTT,0.02% w/v bromophenol blue), boiling for 5 min and running SDS-PAGE to determine the amount and solubility of the GST-fusion protein].

Note:
1. The binding of GST or GST-fusion protein to Glutathione Resin is not affected by 1% Triton X-100, 1% Tween-20, 1% CTAB, 10 mM DTT, 0.03% SDS, or 0.1% NP-40. These chemicals may be used to reduce non-specific binding.
2. If the target GST-fusion protein forms inclusion body (insoluble protein), the inclusion body has to be properly solubilized and refolded prior to purification.

Testdurchführung

Purification of Recombinant GST-Fusion Protein
1. Completely resuspend the Glutathione Resin by gently shaking the vial.
2. Transfer an appropriate amount of slurry to a disposable column (included in Kit L00207 and L00208). Usually 1 ml settled resin (2 ml 50% slurry) can bind more than 6 mg horse liver GST protein.
3. Wash the Glutathione Resin with 10×bed volumes of cold (4°C) 1×PBS.

  1. Apply clear solution (sonicate, etc) containing GST-fusion protein in cold 1×PBS to the equilibrated column with the flow rate at 10-15 cm/h.
    5. Add 1×PBS to wash the column just after all the protein solution get into the column, use 20×bed volumes of PBS for wash. Protease inhibitors such as PMSF are better added to wash solution to inhibit protease activity.
    6. Elute the fusion protein with 10-15×bed volumes of freshly made 10 mM glutathione elution buffer (0.154 g of reduced glutathione dissolved in 50 ml of 50 mM Tris-HCl, pH 8.0.).
    7. Monitor elution of the fusion protein using absorbance readings at 280 nm.
    8. Aliquot 10-20 μl supernatant containing GST-fusion protein, flow-through, wash and the eluted protein, respectively, and analyze all the samples by running SDS-PAGE to confirm the presence of the target protein.
    9. Pool eluted fractions containing target protein. Remove free glutathione by dialysis at 4°C against a buffer of choice or by using a G15 Sephadex desalt column.
Beschränkungen Nur für Forschungszwecke einsetzbar
Format Liquid
Buffer 1X PBS containing 20% ethanol
Lagerung 2-8 °C
Informationen zur Lagerung For long-term storage, the resin should be stored in 1×PBS containing 20% ethanol at 2 - 8°C.
Haltbarkeit 18 months
Produkt verwendet in: Zhang, Zou, Ma, Muhammad, Li, Jiang: "Identification of Mycoplasma suis MSG1 interaction proteins on porcine erythrocytes." in: Archives of microbiology, Vol. 197, Issue 2, pp. 277-83, 2015 (PubMed).

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