1. Take E. coli cell pellet and dilute 20X with lysis buffer in a sealable bottle (i.e. use 20 mL lysis buffer per gram of wet cells). Disperse cells in the solution (mild mixing).

2. Add PMSF (phenylmethylsulfonyl fluoride- a poison!) from a 20 mg/ml stock solution in isopropanol (this can be stored indefinitely in the freezer) to a concentration of 20 mgs per 100 ml of sup (1.1 mM). PMSF is a protease inhibitor which will help keep protein from getting chewed up.

3. Add the following:

lysozyme powder 0.2 mg/ml

powdered DNase and RNase 0.02 mg/ml each

MgAcetate to 5 mM from a 500 mM stock (stock: 11 g/100 ml)

The RNA and DNA in E. coli tend to form thick suspensions.

RNase and DNase will break up this goop.

Seal the container and incubate for ½ hour at room temperature with tumbling

(do not mix with stir bar).

 

4. Tip sonicate at 50% power, 50% duty cycle for 5 minutes (5 sec on, 5 sec off). Place your sample in an ice water bath during sonication.

5. If indicated for your future analyses of pure protein, add dithiothreitol (DTT) to a concentration of 0.5 mM (10 mgs per 100 ml). DTT is a reducing agent which will help keep the Cys thiol groups from getting oxidized. If more than 0.5 mM DTT is added, new nickel resin, not regenerated, must be used.

6. If you wish to stop at this point, lysate can now be divided up into „T40 ml portions in 50 ml Falcon tubes. These can then be frozen in liquid nitrogen and stored in the -80 ¢XC freezer. Note: it can be frozen and stored at this point (before adding detergent), but not after adding detergent.