You can increase wash steps. How do you elute your protein from the GST Sepharose column? I cutted the protein with precission enzyme and I obtain very clean protein of interest
Hopefully you included a protease site so you can cleave off the GST tag after your first purification. Then run it through the column- your protein will not be in the supe, and the extra bands would be on the column. If no protease site maybe preparative PAGE to purify by size.
We have a PreScission Protease site. Unfortunately, even after digestion, I have some impurities coming out with my digested fragment. In some cases, I have observed the cleaved tag coming out as well in the same fraction as the cleaved protein.
Anna: Could you elaborate on which wash steps could be increased? I meant to say, do you advise increasing the wash after loading the protein?
yes, I meant to increase the wasing steps after loading the protein on the column. The protocol I used for recombinant proteins fused with GST-tag expressed in E.coli was as follows:
For protein purification, cell pellet from 250 ml of culture was resuspended in 4 ml of 1X PBS and sonicated on ice. The lysate was centrifuged for 30 min at 12000g and the supernatant mixed with 0.5 ml of Glutathione Sepharose 4B resin (Amersham), previously equilibrated with ten volumes of the same buffer.
The resin was then packed on column by gravity and unbound fraction recovered. The column was washed extensively with PBS monitoring proteins elution spectrophotometrically; when the flow-through reached an OD280 near 0, Digestion Buffer (50 mM Tris HCl, pH 7.0; 150 mM NaCl) was applied to the column. After equilibration of the resin in this buffer, PreScission Protease (Amersham) was added.For me worked well ON digestion with Precission at 4°C.
Are you using the same buffers?
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