On Sat, 20 Jan 2001, John Lum wrote:
> Hi there,
>> > Do you know when the degradation is happening? I think degradation is
> > quite common for GST fusions. For some fusions I made, the degradation
> > only happened during cell lysis- as seen in crude soluble extracts, but
> > not in total protein from boiled cells. Try different extraction
> > conditions if this is the case. I found degradation to be much reduced
> > when 1 mM DTT was included and Triton-X100 left out of the lysis buffer.
>> I'm analysing the total protein from boiled cells. When I use the GST
> antibody for western, I found multiple bands of smaller size.
>>>In that case, the problem is with the induction. Try changing time of
induction (less may give more full-length), temperature (lower?) and
concentration of IPTG. The last one can work very well with the new TUNER
cells from Novagen (No affil.) I got maximal induction from one protein at
40 micromolar IPTG, less at higher concentrations.
Hope this helps,