Materials and Reagents
1. Add BugBuster TM protein extraction reagent at a 5/1 ratio (i.e. 5 mL of reagent for every 1 g of wet cell pellet). If your pellet weighs less than 1 gram, please add 5 mL of protein extraction reagent.
2. Re-suspend the cell pellet in the protein extraction reagent (please ensure that your cell pellet is fully re-suspended in solution, not just detached off the side of the tube). You can re-suspend the pellet by pipetting it up and down in the solution added.
3. Incubate the re-suspended cells at room temperature for 20 minutes in the shaker at low speed.
4. Combine all re-suspended cell suspensions/ lab mentor into one, 50 mL polypropylene tube (choose any one tube from your lab mentor team). To pellet the cellular debris, spin the lysed cells at 16,000 x g for 30 minutes at 4 ˚C in the refrigerated floor centrifuge.
5. Each lab pair: remove your specific volume of supernatant from the combined tube and transfer the supernatant into one, 50 mL Conical tube. Make sure you label the tube appropriately.
6. Place cell lysate on ice. Proceed to the column chromatography step (in practice we ask our students to prepare the column while lysing cells).