Hi,
I am purifying a protein having GST tag. I am using 20mM tris pH 7.5, 200mM NaCl, 5mMDTT and 5% glycerol as the equlibration buffer. Column is of 2ml bed volume (I have hardly 3mg of my protein). Protein is not completely binding to the column and nearly 30% I can find in the flow through. Please suggest me how to increase the binding capacity of the protein.
Thanks in advance..
Replace 5mM DTT (competes with GSH) with 1 mM BME, include 1 mM EDTA (removes metal inhibitor of GST). During sonication do not include detergent. After sonication add 1 mM Triton X 100, mix by inversion 6 times and take supernatant for affinity purification.
Replace 5mM DTT (competes with GSH) with 1 mM BME, include 1 mM EDTA (removes metal inhibitor of GST). During sonication do not include detergent. After sonication add 1 mM Triton X 100, mix by inversion 6 times and take supernatant for affinity purification.
Hi there,
As the capacity of binding is around 25mg of GST per mL of beads (for GE glutathion sepharose 4B), it's hard to believe you are saturating the binding sites of your 2mL column...
Are you sure the unbound is actually properly folded? If it's not it might explain the protein is not binding to the column. What happens if you load the unbound on an other GSH-column? If you haven't tried, I suggest you to do it : if a significant amount is binding it means the unboud has the capacity to bind the column and then it's worth trying to improve the binding, if not the protein has no GSH binding capacity...
The efficiency of binding is flow rate dependent so you can try to decrease it during binding or even to do the binding in batch.
Hi, Manjula,
The binding to GSH beads depends also on the size of your protein. The 25-30mg/ml is only the binding capacity of app. 30kDa protein. If you have larger proteins, you have to calculated less bound protein. I am working with complexes of 300-600kDa, where one of the proteins has GST tag and the binding capacity is 0.5 to 1 mg of the complex /ml resin! So,take more beads to bind more protein. The resin is very specific for the GST, so do't worry to have more impurities when washing well with lysis buffer.
But first check, if the protein in the flow through still binds to GSH beads.
Good luck
Jana
Hi I did purification of GST-tagged protein for several times. My protein bound to the resin loosely, so I use more resin, and also I use the PBS buffer pH7.3 ( which was suggested by protocol) instead of Tris. It came out nicely.
The protocol also suggest that you could mix your supernatant of extraction with GST-resin and shake 1 hour at room temperature, then reload to the column.
Good Luck
Weiping
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