On Fri, 19 Jan 2001, John Lum wrote:
> Hi there,
>> I'm expression a 120kDa protein as a GST-fused protein in E.coli
> BL21. However, my western blot indicates multiple bands of smaller size
> (25-70kDa), but the expected 120kDa protein is abscent. Can anyone give
> me some advice on this matter?
>>>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.
Hope this helps,
University of Leicester
Leicester LE1 7RH
Email: pjs14 at le.ac.uk