Purification of GST Fused Proteins
|Author: Chia Jin Ngee|
|Source: Contributed by Chia Jin Ngee|
|Date Added: Tue May 14 2002|
|Date Modified: Sat Feb 21 2009|
|Abstract: Many people have vented out frustration over insoluble GST-fused proteins. This is a protocol for enzymatically active soluble GST-fused proteins. All GST-fused proteins are rendered soluble with this technique though enzyme activitiy can range from 30-90%.|
Materials and Reagents
Pour the lysate to 1 ml bed of prepared Glutathione Sepharose in PBS. Incubate at room temperature for 30 min to 1 hr with agitation.
To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml tube. Fill to 50 ml with PBS, invert tube a few times. Centrifuge to 2000 rpm on a swing bucket centrifuge then switch off. Carefully suck off PBS and resuspend beads with 1 ml of PBS.
Wash the beads with 3 X 50 ml of PBS. Finally resuspend in 5 ml of PBS. Pour to a dispo-column. Wash the 50-ml conical tube with an additional 5 ml of PBS. Pool with the first 5 ml in the dispo-column.
To wash, use the same centrifugation technique for preparing the beads. When transferring beads to column, do not pipette but pour. The beads tend to stick to pipette tips.
If desired, elute with 10 x 1 ml fractions of Elution Buffer. Determine desired fractions with SDS PAGE.
Frangioni and Neel.Anal. Biochem. 210, 179-187 (1993)