Troubleshooting protein expression - GST tag - (Jan/11/2007 )
I am attempting to express a recombinant GST tagged protein. Following a mini-scale induction and western with the protein-specific antibody I am seeing what I presume is my product at approximately the correct size. There is not a lot there although I haven't quantified the total protein in the samples as yet. There is also a smaller product present in all my samples, including the uninduced bacterial pellet. My GST blot was overexposed so I am not sure what is carrying a GST tag. When I tried in another experiment to elute my protein from beads I got a band at the about the same size as the 'smaller' band in my westerns. I'm not sure what is going on?
*Does the smaller band represent some basal level of protein expression?
*There seems to be a bit more of the correct sized protein in my pellet (ie insoluble fraction) = inclusion bodies?
*Should I try another type of bacteria for the expression. At the moment I'm using bog standard BL21.
Thanks for any advice. I've had either works or doesn't work before.....not 'sort of' works!
where do you see the smaller band? western? maybe your protein is cleaved.....and this is shorter fragment of your protein.
if you see enough protein in the soluble fraction dont bother about something present in the pellet. just go on.
first try to figure out what is the problem. BL21 is standard for protein expression, so first check what is the problem and then think about changing it.
if you are getting enough protein in your soluble fraction, then you dont have inclusion body problem. if you have some small fragment then maybe you have degradation problem.
Thanks. Yes, I can see the smaller fragment by Western - I can't make out anything obvious in a coomasie gel. I've done the GST blot now and there is a smaller band in all samples including uninduced and then a higher band which I presume is my protein in the induced samples. In induced samples there is a LOT of smaller bands being detected by the GST antibody so I presume there is some degredation problem. Off to try and troubleshoot that.
I only asked about the bugs because in the past we've used codon plus ones if we couldn't express in normal BL21s.
why there are small bands in uninduced samples? there is a phenomenon called "leak expression". never faced it and dont know if it's related. maybe try to read about it.