Hi,
I've read an application sheet, there was a statement was given below;
''This protocol describes the generation of a cleared lysate from an E. coli cell pellet and the subsequent purification of GST-tagged proteins under native conditions using Glutathione Agarose. ''
The elution buffer recipe was; Tris base, pH 7.4, NaCl 150 mM, Triton X-100 0.1% (v/v), Reduced glutathione 50 mM, DTT 1 mM.
In the case of detergent and reducing agent availability during purification, is it possible to keep the target protein in its native form?
I wonder, with the purposed elution buffer (that includes DTT) how can the GST tagged protein be eluted in native form?
Thanks in advance...
Hi there,
0.1% of Triton X-100 is not denaturing for most proteins.
Cytosolic expression in E. coli is suitable for proteins in which disuphide bonds formation is not essential for stability (cytosol is highly reducing). Therefore these proteins will also be able to be purified in the presence of reducing agent.
In addition to Dominique's answer, in most cases Triton X-100 is not essential to elute the protein from glutathione agarose. Its purpose is to prevent non-specific sticking of the protein to the resin in case it should be a problem, but it is better to include it in the wash buffer to eliminate non-specific sticking of contaminating proteins. DTT is added to maintain glutathione in the reduced form but in case the desired protein cannot tolerate it, it could probably be left out if reduced glutathione is added to degassed elution buffer.
Pierre Béguin and Dominique Liger thanks for your answers. They were enough to solve my confusion.
- Badges
- Science method