Me and my PI are doing a simple protein gel prior to a normal western to see if our protein was successfully expressed in BL21. The following protocol is a rough outline of what we hope to use on 10 mL volumes spun down to pellet before committing the time and reagents to a proper purification and western blot to see if we have protein. I was hoping some people could look it over before hand and over any advice/their opinions.
1. Resuspend pellet in an 8M urea solution (with protease inhibitor) and let sit on ice for 30 minutes
2. Spin down 5 minutes at 10k rpm
3. Discard supernatant
4. resuspend in 8M urea solution and add 20uL Ni resin (transfer 1mL to eppendorf tubes)
5. Let sit for 30 minutes on ice
6. Spin down and remove supernatant
7. Add 8M urea and wash 2x
8. load 20uL beads + loading buffer into gel and run.
Note: we are just hoping that this will make it easier to see out target bands rather than simply lysing the pellets with 8M urea solution and running it on a gel as there might be too much background from the bacterial proteins.