DE52 med ium. column Gradient maker. UV-monitor and fractioner Method I Thaw the cell pellet and vortex 2 Add 0. 4ml 100 mM PMSF and sonicate(400kw, 4s-6s 50 cycle* 5) 3 Centrifuge 15000 rpm, 30 minutes to pellet debris 4 Transfer supernatant to a 50 ml conicale tube and d iscard the pellet 5 The supernatant dilute to 50 ml with Buffer A and then load to de52 ion-exchange columns(20mD), which was pre-equibrated with 100ml Buffer A. And then wash the unbound proteins with 100 ml Buffer A 6 Elute the protein with a linear gradient: 200ml buffer a plus 200ml buffer B 2ml/min 6ml each fraction 7 Run 15% SDS-PAGE to determine the HSP16.3 peak Desalting by dialysis I Preparation of the dialysis tube Cut the tube in a suitable length(20-30 cm) Boil the tube in solution containing 10 mM NahCo3 for a few minutes Boil the tube in solution containing 10 mM EDTA for a few minutes Rasin the tube with de-ion water 2 Pool the HSP16. 3 peak and dialysis the Sample against 1000ml Buffer A for more han 6hours- DE52 medium , column ,Gradient maker, UV-monitor and Fractioner - Tips Method: 1 Thaw the cell pellet and vortex . 2 Add 0.4ml 100 mM PMSF and sonicate (400kw, 4s-6s 50 cycle* 5 ) 3 Centrifuge 15000 rpm, 30 minutes to pellet debris 4 Transfer supernatant to a 50 ml conicale tube and discard the pellet. 5 The supernatant dilute to 50 ml with Buffer A and then load to DE52 ion-exchange columns (20ml), which was pre-equibrated with 100ml Buffer A. And then wash the unbound proteins with 100 ml Buffer A. 6 Elute the protein with a linear gradient : 200ml buffer A plus 200ml buffer B, 2ml/min, 6ml each fraction. 7 Run 15% SDS-PAGE to determine the HSP16.3 peak. Desalting by dialysis 1 Preparation of the dialysis tube Cut the tube in a suitable length (20-30 cm) Boil the tube in solution containing 10 mM NaHCO3 for a few minutes. Boil the tube in solution containing 10 mM EDTA for a few minutes. Rasin the tube with de-ion water 2 Pool the HSP16.3 peak and dialysis the Sample against 1000ml Buffer A for more than 6hours