Expression of a 8-kDa protein in E coli

[TR]

Hallo everybody,

We are trying to express in E. coli a small protein (77 aa, 8090 Da),
using the pRSET system and BL21 pLysS. We can't see any band of the
expected size in our PAGEs (20% acrylamide), but we fear that such a
small protein will disappear from the gel during staining (with
colloidal coomassie). We are trying a different staining procedure in
which small proteins are fixed before staining, described in this
group a long time ago (see below). Any other suggestion to be able to
'see' this small recombinant protein?
Thank you in advance,
CG



Quoted from Frank O. Fackelmayer (Fri Dec 7 10:20:53 EST 2001):
>> in many cases, small proteins are not fixed well in the gel, and readily
>> diffuse out during the staining process. We had the same problem, until
>> we switched to a different formulation of the coomassie staining
>> solution. Using one with formaldehyde provides a much better fixation,
>> and a much higher sensitivity for small proteins. The formulation is
>> described in Steck et al, 1980, Anal. Biochem. 107:21-24.
>>
>> Staining solution:
>> 18ml EtOH, 42ml Water, 10ml formaldehyde (from 37% stock), 0.08g Coomassie
>> (as always with coomassie solutions, do NOT reuse!)
>>
>> destaining solution:
>> 250ml EtOH, 750ml water, 10ml formaldehyde
>>
>> staining takes approximately the double time, we used 1h for minigels.
>> Replace with destaining solution as usual, until bands are well visible.
>> In contrast to normal coomassie staining procedures, the sensitivity
>> increases quite significantly when you store the gel in destaining
>> solution plus 1/10 volume staining solution, overnight in the fridge.