GST fusion protein preparation protocol and troubleshooting - (Apr/03/2007 )
I require soem help in my experiment.. Did a few round already, cos the first round, my protein was not pure ie cross contamination between V8-GST adn GST protein. 2nd round was low yield.. 3rd round (the most recent) the protein sort of degraded a little bit.
But I made sure not to cross contaminate by using loads of pipettes.. And I made sure to put my protein samples on ice all the time. And centrifuging at 4 degrees when i need to wash off excess protein bound to beads. What could cause all possible errors?
Could soemone pls help with troubleshooting, ie suggest possible steps in which i could hv gone wrong??
these are the steps i really paid attention to:
1. prevent cross-contamination by using different pipettes
2. put samples on ice all the time
3. use fresh elution buffer and lysis buffer
4. sonicate so that cell culture become clear
sonication is used to lyse cells and to break DNA but in excess can degrate your protein and multiple bands can appear.
you should use protease inhibitors before cell lysis but dont use EDTA if you plan to use DNase.
detergents, salt, and DTT prevent unspecific purification.