Problem with protein expression
peter_frankde at yahoo.de
Thu Sep 30 15:02:36 EST 2004
Kyle Legate wrote:
>Peter Frank wrote:
>> I have some problems expressing a His-tagged protein. I cloned the
>> sequence in-frame into the pET-20b(+) expression vector, which I used
>> to transform E.coli BL21 (DE3) cells with. I thoroughly checked that
>> insert and His-tag are in-frame. I grew the culture at 30 °C, and
>> although it grew slowly, it did grow continuously as monitored by OD
>> measurements. So, I assume the protein to be expressed is non-toxic. I
>> lysed the cells completely. I purified some lysate using Ni-NTA
>> magnetic agarose beads, which should highly enrich the His-tagged
>> protein. I ran several SDS-PAGE gels with purified and unpurified
>> lysates (with non-reducing and reducing sample buffer). The
>> silver-stained gel did not show any band with significantly increased
>> intensity (but then again there were many other bands of course that
>> may obscure this).
>This may be a dumb question, but did you induce your cells? You didn't
>mention this step.
Forgot to mention it. I did induce my cells with IPTG at a final
concentration of 0.4 mM.
>There is an E. coli protein that reacts with the anti-His
>antibody (at around 60 kDa?), and you may be detecting this in your Western.
The band I see is around 30 kDa. Don't know what it is.
>It's a good idea to compare some induced E. coli lysed directly with SDS
>loading buffer with the lysate you intend to use for purification to
>determine if your protein is soluble.
Does SDS loading buffer lyse absolutely everything?
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