GST-fusion elution problem

D.K. dk at no.email.thankstospam.net
Thu Sep 27 10:10:20 EST 2001


maeda at ims.u-tokyo.ac.jp ("MAEDA, Tatsuya,  Ph.D.") wrote:
>Dear All,
>
>We have expressed a GST-fusion protein in bacteria.  Fortunately, the
>protein was present in the supernatant following centrifugation of the
>lysate.  Using glutathione beads, the protein was successfully recovered
>from the supernatant.  And here comes a problem.  The protein resists to be
>eluted from the beads.  We have tried several elution solutions: 10mM
>glutathione 50mM Tris-Cl pH8.0, higher glutathione conc.(up to 50mM),
>higher pH(pH9.6), detergent(0.2% Triton X-100), and higher salt conc.(up to
>500mM NaCl), but all in vain.  Has anyone come across the same situation
>and does anyone know any remedies?

Strange. No elution at all? 

Check final pH of the solution and leave the sorbent overnight in 50 mM 
glutathione, pH 8.3. Unless you fusion itself binds glutathione even better, 
this should elute. 

DK
 




More information about the Methods mailing list