Hi! I am trying to purify GST tagged GFP. I cloned GFP into the pGEX-2t vector. We used BL21 cells, grew to an OD600 of 0.6, added 1 mM IPTG and grew overnight at room temperature. We harvested the next morning. The protocol that we use (that we received from another lab) involves spinning down the cells, removing supernatant, resuspending in PBS, lysozyme at 37 degrees, heat shock, DNase for 30 mins on nutator and then we try binding to the column (we are purifying using GST spin columns from Thermo as this is for a student lab and we don't need large amounts of protein). Our protocol does not add any protease inhibitors and does not mention the need to work on ice.
Our major issue is that the GST-GFP never binds to the column... it all comes through in the flow through (easy to see as the flow through is glowin). I noticed in the manual for the GST spin column that we were to do the protocol at either room temp or 4 degrees. As we do not have a refrigerated centrifuge we do not have the option of performing all the spins at 4 degrees. Could this be an issue?
The people that we got our protocol from have since switched to using a His tag. They were finding that either during expression or isolation of the protein that the protein was degrading. On a western blot they found GST only at 27 kDa and not at the weight of the fusion protein. The cells glowed following expression so we know that GFP had to be there as well, although perhaps not attached to GST. The way that the construct was created had the promoter, then GST tag and then GFP so we would think they would be transcribed together. Have you heard of any situations where the GST tag detached from the protein of interest? Perhaps by some sort of protease cleavage?
Thanks in advance for any advice!