Team:WashU/Protocols/Celllysis

Cell Lysis and Protein Purification Prep

Buffer:

1. The appropriate amount of tris(hydroxymethyl)aminomethane (Tris) was weighed to create a 50 mM solution. (6.057g/ liter of solution).
2. Similarly, ethylenediaminetetraacetic acid (EDTA) was weighed out to create a 1 mM solution. (0.292g/ liter of solution).
3. The two substances (Tris and EDTA) were dissolved in deionized water. (The solution had some difficulty dissolving and so was placed in a 37°C water bath for a few hours.)
4. Now, the solution was pH-ed using 1 M HCl to a pH of 7.5. (NaOH may be added in the event of an overshoot.)

Lysis:

1. Spin down the cells desired in a microcentrifuge for 1 minute at 12,000RPM. Discard supernatant and resuspend the culture in the buffer above.
2. Sonicate or use some other method to extract proteins.
3. Refer back to the SDS-PAGE protocol to see how to load the gel.