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Glutathione Resin Bead

Pull-Down Glutathione Agarose beads 90 µm
Pubmed (11)
Catalog No. ABIN1536559
$159.47
Plus shipping costs $45.00
10 mL
local_shipping Shipping to: United States
Delivery in 4 to 6 Business Days
  • Application
    Pull-Down Assay (Pull-Down)
    Specificity
    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.
    Characteristics
    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
  • Reagent Preparation

    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.

    Sample Preparation

    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.

    Assay Procedure

    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.
    Restrictions
    For Research Use only
  • Format
    Liquid
    Buffer
    1X PBS containing 20% ethanol
    Storage
    2-8 °C
    Storage Comment
    For long-term storage, the resin should be stored in 1×PBS containing 20% ethanol at 2 - 8°C.
    Expiry Date
    18 months
  • 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).

    Theoret, Uzal, McClane: "Identification and characterization of Clostridium perfringens beta toxin variants with differing trypsin sensitivity and in vitro cytotoxicity activity." in: Infection and immunity, Vol. 83, Issue 4, pp. 1477-86, 2015 (PubMed).

    Sainz-Polo, González, Pastor, Sanz-Aparicio: "Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Paenibacillus barcinonensis xylanase 10C containing the CBM22-1-CBM22-2 tandem." in: Acta crystallographica. Section F, Structural biology communications, Vol. 71, Issue Pt 2, pp. 136-40, 2015 (PubMed).

    Spagnol, Reiling, Kieken, Caplan, Sorgen: "Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain." in: Biomolecular NMR assignments, Vol. 8, Issue 2, pp. 263-7, 2014 (PubMed).

    Smardon, Diab, Tarsio, Diakov, Nasab, West, Kane: "The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast." in: Molecular biology of the cell, Vol. 25, Issue 3, pp. 356-67, 2014 (PubMed).

    Ma, Aslanian, Sun, Jin, Shi, Yates, Hunter: "Identification of small ubiquitin-like modifier substrates with diverse functions using the Xenopus egg extract system." in: Molecular & cellular proteomics : MCP, Vol. 13, Issue 7, pp. 1659-75, 2014 (PubMed).

    Li, Zhu, Li, Ren, Huang, Lu, Fang, Kang: "A novel myeloid differentiation factor 88 homolog, SpMyD88, exhibiting SpToll-binding activity in the mud crab Scylla paramamosain." in: Developmental and comparative immunology, Vol. 39, Issue 4, pp. 313-22, 2013 (PubMed).

    Petrosyan, Ali, Verma, Cheng, Cheng: "Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail." in: The international journal of biochemistry & cell biology, Vol. 44, Issue 7, pp. 1153-65, 2012 (PubMed).

    Ali, Chachadi, Petrosyan, Cheng: "Golgi phosphoprotein 3 determines cell binding properties under dynamic flow by controlling Golgi localization of core 2 N-acetylglucosaminyltransferase 1." in: The Journal of biological chemistry, Vol. 287, Issue 47, pp. 39564-77, 2012 (PubMed).

    Bobba, Ponnaluri, Mukherji, Gutheil: "Microtiter plate-based assay for inhibitors of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus." in: Antimicrobial agents and chemotherapy, Vol. 55, Issue 6, pp. 2783-7, 2011 (PubMed).

    He, Pan, He, Lin, Liao, Zuo, Yuan: "Structural and functional studies of leptins from hibernating and non-hibernating bats." in: General and comparative endocrinology, Vol. 168, Issue 1, pp. 29-35, 2010 (PubMed).

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