[Bacterial cell lysate preparation]
Bacterial cell lysates containing expressed water-soluble GST-fusion proteins can be prepared by many different methods, such as sonication, lysozyme treatment, cell disrupting reagents, etc. We have developed a very effective method to lyse bacterial cells by lysozyme treatment using SureLyse^TM reagent. Water-soluble GST-fusion proteins expressed in bacterial cells can be solubilized by this method for purification by automation on Magtration® 12GC robot.

[I. Preparation of bacterial lysate containing water-soluble GST-fusion proteins using SureLyse^TM reagent]

A. Reagents required

1. SureLyse^TM reagent (Included in Magtration-GST^TM kits)

2. 20 mg/ml lysozyme in ultrapure water. Prepared freshly by users. It can be aliquoted and stored at -20°C for use within one week.

3. 1000 units/ml DNase I in ultrapure water. Prepared by users. It can be aliquoted and store at -20°C for use within one month.

4. Optional but recommended is complete, mini, EDTA-free protease inhibitor cocktail from Roche (Cat. # 04693159001)

Lysozyme and DNase I can be purchased from Sigma, Catalog# L7651 and D5025, respectively.

B. Preparation of bacterial lysate for sample size 1-4 ml pellet (up to10 OD600 units of cells)

1. Determine the O.D.600 for a fresh bacterial culture. An O.D. in the range of 1-2.5 is suitable for the SureLyse^TM reagent treatment.

2. Centrifuge the bacterial culture of 1-4 ml at 10,000 rpm/3 min at 4°C. Discard the supernatant by inverting the tube and tapping it gently on paper towels on a lab bench. The pellets are frozen at -75 °C (±5) for at least 1 h.

3. Thaw the pellets at the room temperature, mix with 50 ul SureLyse^TM reagent. We recommend that the SureLyse^TM reagent protease inhibitors. The mixtures are frozen again at -75 °C (±5) for at least 1 h before use.

4. Thaw the mixture and add 50 µl of 20 mg/ml lysozyme and 5 µl of 1000U/ml DNase I. Rock for 20 min at room temperature.

5. Add 500 µl of SureLyse^TM reagent to the mixture in a 1.5-ml screw cap tube (provided with the kits), and place them in the tip/tube rack of the 12GC robot. We recommend that the SureLyse^TM reagent containing protease inhibitors. The final sample volume is 600 µl.

C. Preparation of bacterial lysate for sample size of 5-20 ml pellet (10-25 OD600 units of cells)

The lysate preparation for the larger sample size of 5-20 ml pellet is essentially the same as for the sample size of 1-4 ml pellet as described above, except that in

Step I.B.3. add 200 µl of SureLyse^TM to the thawed pellet;

Step I.B.4. add 100 µl lysozyme and 20 µl DNase I to the thawed mixture; and

Step I.B.5. add 900 µl of SureLyse^TM reagent to the mixture. The final sample volume is 1200 µl.

[II. Other lysis methods suitable for this kit]

A. Sonication

Because each laboratory has different instrumentation, no protocol is provided for sonication. Several general guidelines for sample preparation for this kit should be followed.

1. The amount of bacterial culture per sample should be similar to the SureLyse^TM method.

2. The sonicated sample has to be transferred into the 1.5 ml screw top tubes included with the kit before placing in the tip/tube rack.

For the 1-4 ml protocol, sample volume is 0.6 ml

For the 5-20 ml protocol, sample volume is 1.2 ml

3. The buffer should be compatible with GST-fusion protein binding to glutathione beads. Phosphate buffered saline containing protease inhibitors is recommended.

B. Cell disruption reagents
1. Check the manufacturer’s documentation for suitability of the reagent for GST-fusion protein purification and for limitations on amount of sample per volume of reagent.

2. Addition of DNaseI to the sample is highly recommended as undigested DNA will interfere with the magnetic separations.

3. The sample has to be transferred into the 1.5 ml screw top tubes included with the kit before placing in the tip/tube rack.

For the 1-4 ml protocol, sample volume is 0.6 ml

For the 5-20 ml protocol, sample volume is 1.2 ml

4. Addition of protease inhibitors is recommended. 2