Beads pull down only free GST - (Mar/31/2015 )

Instead of a dot blot, do a full blot running whole cells immediately after expression. This will tell you whether the cells are producing full length protein. If they are, then you know something is truncating your protein during the purification. If they are not, then construct and expression needs to be optimized. In other words, running a full blot will guide you where to focus your troubleshooting.

Hey you, thanks for your answers.

I already loaded the stuff on a sds gel and yes it is the full length protein there. Also sent it to mass spec. The only problem is, that it seems that the GST is cleaved from the protein right during the expression.....

The beads are new, just bought them for these purposes.

it may be a chore but you may want to re-engineer your insert with a different linker that may be less susceptible to cleavage.

or you may be able to change your host to one that won't cleave the linker you're using.

Is there any particular reason you have to use a GST tag?  

I've found many times that published/lab-standard methods are not always optimized. You'd be surprised that if you ask people why they express and purify their protein a particular way, more often than not it's because "that's what the previous grad student did", or "that's what the paper says to do", or "this what someone told me to do"....rarely is it because "this method has been optimized and it works much better than these other methods, as you can see from this data in my notebook".

I've had a lot of luck expressing proteins with cleavable SUMO and GFP tags.

If you want to get fancy, you could double tag your protein (eg Nterm His tag, Cterm strep tag) so that you only purify full length, non-proteolyzed protein.

Thanks again for your answers.

The main reason i am using GST tag is because we had this construct and it worked in former times. In our lab there is a microbiologist who helped me to optimize the culturing for the auto induction bcause the iptg induction didn't work for me, but also he doesn't know why it is cleaved during expression.

The vector itself/Protein expression with GST tag has a manual or handbook from GE healthcare which is very detailed but also there they don't write anything about it.

Another point of using GST tag is, that i wanted to bind the GST-protein to beads for a pulldown.

Of course i could buy another vector and clone it again but i am not very lucky with cloning