Good thing takes time

2. Preparation of loading samples

- thaw cells on ice or RT (10 g of cells in 10 ml of Buffer I)

- add 5 ml of acid-washed glassbeads and protease inhibitors cocktail

- vortex 20'' and cool mixture at -5oC for 40'' as one cycle, repeat vortex cycle for 10 min.

- spin lysate at 7700g for 10 min

- transfer supernant to a clean tube (about 8 mL)

- dilute the supernant by Dilution Buffer (ratio 1:4)

- high salt-PEG precipitation for 30 min on ice by mixing

- spin at 10-12,000 Xg for 60 min

- about 30 ml of supernant is the sample for loading on HAP column.


## Buffer I:
50mM Tris pH 7.7/ 1mM EDTA/ 1mM EGTA/10 % glycerol/ 25 mM sodium fluoride/ 1mM sodium bisulfite/ 1mM PMSF/ 1mM b-mercaptoethanol/ 1mM benzamidine/ 5 ug/ml leupeptin/ 5ug/ml pepstatin.

## Dilution Buffer:
30 mM KPi (3 mL 2M KPi)
2mM MgCl2 (0.4mL 1M MgCl2)
1mM PMSF ( 2mL 0.1 M PMSF)
1 mM Beta-Mercaptoethanol (-.014 mL 14.2M beta-ME)
1mM DTT ( 0.2 mL 1M DTT)
10 % glycerol ( 20 mL gylcerol)
1M NaCl ( 50 mL 5M NaCl)
5ug/ml leupeptin (0.5mL 2 mg/mL)
5ug/ml pepstatin A (0.5mL 2mg/mL)
dH2O 103.4mL

# Dialysis Buffer: (4 Liter)
Kpi pH 7.0 30mM 60mL 2M
Glycerol 50% 2 liters
DTT 2mM 8mL 1M
EDTA 0.5mM 4mL 0.5M
PMSF 1mM 4mL