Glutathione Affinity Superflow 4

Glutathione Affinity Superflow 4 is manufactured by the optimized immobilization of high-grade glutathione to Sterogene`s trademarked Superflow 4 matrix. Glutathione/GST purification has proven to be a simple, effective method of purifying recombinant proteins expressed in the pGEX vector. Once bound to the resin, the GST-tagged protein can be eluted with glutathione, or it can be cleaved from the protein while still on the column. This results in the elution of the GST-free recombinant protein.

This media provides an effective and efficient one-step purification for a variety of different expression systems. Along with the high binding capacity for GST-tagged proteins, Glutathione Affinity Superflow 4 shows low non-specific binding and is comparable in performance to the leading immobilized glutathione resins available.

cGMP manufacturing under ISO 9001:2000; Regulatory Support File available
-DMF with USFDA

Product Code: 37009SF4
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Table 1. Resin Characteristics

Bead Material Agarose
Bead Percentage 4%
Bead Size60-160 µm
Flow Rate1>20 mL/min (>680 cm/hr) @ 25oC
AT3 Binding >6 mg/mL
pH Stability22-14
Storage Temperature2-8o C
Storage Buffer 20% Ethanol
FormSlurry
Chemical Stability2Stable in all commonly used aqueous solutions and buffers.
Physical Stability2Negligible volume variation due to changes in pH or ionic strength.

1Linear flow rate = volumetric flow rate (cm3/h)/column cross-sectional area (cm2)

2Data refer to the coupled product, provided that the ligand can withstand the pH or chemical environment. Please note the following: pH stability, long term refers to the pH interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.

Instructions for Use

Glutathione Affinity Superflow 4 is a resin used for the purification of a variety of different Glutathione (GST) tagged proteins including: Glutathione S-transferases, Glutathione-dependent proteins, and recombinant derivates of Glutathione S-transferases.
Glutathione Affinity Superflow 4 purification requires some degree of method development for optimization. The protocol below is meant as an example for binding GST tagged proteins.

Protocol:
Reagents:
Wash buffer: PBS (1X)
Binding buffer PBS (150 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO5), pH 7.35.
Elution buffer: 50mM Tris-HCl, 10mM reduced glutathione, pH = 8.0.

*** If necessary, the addition of dithiothreitol (DTT) to either the wash and/or elution buffer can reduce the risk of oxidation of free thiol (-SH) groups on GST. This dimerization of the GST-tagged proteins can result in precipitation and lower yields. It is recommended that 1-20 mM DTT be used. ***

Purification of GST Tagged Proteins
1. Pour the desired amount of Glutathione Affinity Superflow 4 in a Buchner funnel.
2. Wash resin with 10 bed volumes (BV) of wash buffer to remove all residual 20% ethanol.
3. Suction dry resin and remove to a designated container.
4. Add the cell lysate to the resin.
5. Rock/mix for 30-45 minutes at room temperature to ensure complete equilibration.
6. Remove sample and suction dry resin. Make sure to collect flow-through for further analysis.
7. To the resin, add 3 BV of wash buffer and mix for 3-5 minutes at room temperature.
8. Repeat steps 6-7 three additional times and collect fractions. This will result in the removal of any unbound material.
9. Elute the bound protein by adding elution buffer in a 1:1 ratio to Glutathione Affinity Superflow 4 resin.
10. Rock/mix for 30 minutes at room temperature to ensure complete equilibration.
11. Remove sample and suction dry resin. Make sure to collect flow-through for further analysis.
12. Repeat steps 9-11 three additional times and collect fractions.
13. Analyze each fraction for purified protein by measuring absorbance at A280.
14. Collect and pool fractions of purified protein together.

Method for Cleaning/Regeneration:
1. Wash the resin with 3-5 BV of a solution containing: 0.1M Tris-HCl 50mM Tris, 0.5M NaCl, pH 8.5.
2. Wash the resin with 3-5 BV of DI water.
3. Wash the resin with 3-5 BV of 0.1M Sodium Acetate, 1M NaCl, pH 4.5.
4. Wash the resin with 3-5 BV of DI water.
5. Wash the resin with 3-5 BV of wash buffer to re-equilibrate.

For technical service email info@sterogene.com or call (760) 929-0455

To Download Instructions for use:

INST 37009SF4 Glutathione Affinity Superflow 4

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