Glutathione Agarose

1. Recommended Buffers

Binding/Wash Buffer, Elution Buffer.

2. Load of Glutathione Agarose

(1) Mix the slurry by gently inverting the bottle several times to completely suspend the agarose.

(2) Transfer an appropriate-sized slurry to the column and allow the agarose to settle down and the storage buffer to drain from the column.

(3) Equilibrate the column with 3× bed volumes of ultrapure water

(4) Close the bottom outlet of the column

3. Procedure

(1) Equilibrate the column of agarose with 5× bed volumes of Binding/Wash Buffer.

(2) Apply appropriate-sized sample to the column. If the sample contains insoluble matter, centrifuge or filter (0.22 μm or 0.45 μm filter) before use.

Note: Binding capacity is flow rate-and protein-dependent. Higher flow rates will decrease production time, but may result in losing a small portion of the target protein.

(3) Collect and save the flow-through for analysis.

(4) Wash the column with approximately 10-15× bed volumes of Binding/Wash Buffer or until the absorbance is stable. If desired, save supernatant for downstream analysis.

(5) Elute with approximately 5-10× bed volumes of Elution Buffer, collect fractions.

(6) Analyze the target protein by SDS-PAGE, along with fractions collected from different steps if necessary

4. Cleaning-in Place, CIP

(1) To remove precipitated or denatured proteins, wash the column with 2× bed volumes of 6 M guanidine hydrochloride followed by 5× bed volumes of PBS (pH 7.4) washed.

(2) To remove hydrophobic substances, wash the column with 3-4× bed volumes of 70% ethanol or 2× bed volumes of 1% Triton X-100 followed by 5× bed volumes of PBS (pH 7.4) washed.

(3) Store agarose in 20% ethanol at 2-8ºC.