Solubility Screening

Preparation:

1. Label 1.5ml Eppendorf tubes for each sample. 
2. Take samples out of the freezer. They will have time to thaw while you make the lysis buffer. 
3. Take the 2x SDS-PAGE sample buffer out of the freezer to thaw. 
4. Place beaker of water on heat block to begin warming/boiling while you are preparing samples. 
5. Take appropriate number of gels out of fridge to warm to room temperature. (Each gel has 16 wells, 1 well is the Low Molecular Weight, 15 wells are left for samples. If you have 30 samples, you will need two gels.)

Bacterial Lysis

1. Prepare lysis buffer (sufficient for 25 samples) by adding 20 l of Sigma Bacterial Protease Inhibitors to 5 ml of 50 mM Sodium phosphate, pH 8.0, 300 mM NaCl. Add 20 l of Epicenter or Novagen recombinant stabilized T4 lysozyme solution, 5 l of Benzonase, and mix. OR use table below to make enough lysis buffer for your number of samples.
   
   

   Reagents	2mL	3mL	4mL	5mL	6mL	7mL	8mL	9mL	10mL	11mL
   H2O	~2mL	~3mL	~4mL	~5mL	~6mL	~7mL	~8mL	~9mL	~10mL	~11mL
   1M Na2PO4	100 µl	150 µl	200 µl	250 µl	300 µl	350 µl	400 µl	450 µl	500 µl	550 µl
   5M NaCl	120 µl	180 µl	240 µl	300 µl	360 µl	420 µl	480 µl	540 µl	600 µl	660 µl
   PI	12 µl	18 µl	24 µl	30 µl	36 µl	42 µl	48 µl	54 µl	60 µl	66 µl
   Lz	8 µl	12 µl	16 µl	20 µl	24 µl	28 µl	32 µl	36 µl	40 µl	44 µl
   Bz	2 µl	3 µl	4 µl	5 µl	6 µl	7 µl	8 µl	9 µl	10 µl	11 µl

   
   PI: Protease Inhibitor Cocktail (Sigma)             **See Lysis Buffer for Solubility Screening for complete chart. 
   Lz: rLyzozyme
   Bz: Bezonase
   
   
2. Lyse the bacteria by suspension of the pellets in 160 l lysis buffer and incubation at room temperature for 5 minutes. During the incubation, vortex the samples 1-2 times. 
3. Transfer the suspension to labeled 1.5 ml microcentrifuge tubes and centrifuge at 12,000 x g at (4°C) for 6 min. 
4. After centrifugation, remove 60 l of the supernatant and add to 50 l of 2x SDS-PAGE sample buffer. After boiling for 3-4 minutes, use 12-15 l for analysis by denaturing PAGE. 
5. Stain using Invitrogen Simply Blue Stain or Coomassie Blue Stain. (See Staining Protocol for proper procedure)