u/Antique_Magician_163

Hello, for some background I am an undergrad who had a single summer internship in a lab focusing mainly on protein purification, and was asked back for a hired summer position this year. My mentor is a post-doc who is slumped with work for a manuscript and I do almost all the manual work for purification. Generally I just follow protocol, but we started a new project with a new batch of proteases.

Currently, our lysing process is not working as it should be. We create our lysis buffer and then manual lyse via sonication, but the solution remains pretty opaque, and when clarifying the lysate, the pellet is much bigger than we want. Ni-NTA Chromatography and running a coomassie gel confirms most of the protein is stuck in the pellet. 

It’s possible that the protein is just insoluble or whatever else, but I’m really not sure. Our project is robust so my mentor is encouraging me just to move on and we’ll revisit, but it’s happened twice now and I’d like to help. Any ideas on how to optimize lysis would be helpful. 

For our current process: We are working with proteases and the current lysis buffer consists of 20mM Tris buffer, 100mM NaCl, 5% glycerol, 5mM BME. - We also induce secondary culture at 0.6-0.8OD with 1M IPTG and leave overnight at 18 degrees celsius. Idk if any of that helps. If more info is needed to help, please lmk!

Hello, for some background I am an undergrad who had a single summer internship in a lab focusing mainly on protein purification, and was asked back for a hired summer position this year. My mentor is a post-doc who is slumped with work for a manuscript and I do almost all the manual work for purification. Generally I just follow protocol, but we started a new project with a new batch of proteases.

Currently, our lysing process is not working as it should be. We create our lysis buffer and then manual lyse via sonication, but the solution remains pretty opaque, and when clarifying the lysate, the pellet is much bigger than we want. Ni-NTA Chromatography and running a coomassie gel confirms most of the protein is stuck in the pellet. 

It’s possible that the protein is just insoluble or whatever else, but I’m really not sure. Our project is robust so my mentor is encouraging me just to move on and we’ll revisit, but it’s happened twice now and I’d like to help. Any ideas on how to optimize lysis would be helpful. 

For our current process: We are working with proteases and the current lysis buffer consists of 20mM Tris buffer, 100mM NaCl, 5% glycerol, 5mM BME. - We also induce secondary culture at 0.6-0.8OD with 1M IPTG and leave overnight at 18 degrees celsius. Idk if any of that helps. If more info is needed to help, please lmk!