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4.

PROCEDURE: a) BUFFER COMPOSITION PREPARATION i. Wash Buffer (1L) 1) 50 mM NaHPO (MW=137.99 g/mol) 2) 300 mM NaCl (MW=58.44 g/mol) 3) 20 mM imidazole (MW=68.08 g/mol) 4) Adjust pH using NaOH ii. Elution Buffer (1L) 1) 50 mM NaHPO 2) 300 mM NaCl 3) 250 mM imidazole 4) Adjust pH using NaOH b) FPLC PROCEDURE: 1) The computer was setup 2) The internal compartment have been washed with 20% ethanol by setting pump wash clarifier by click on execute. 3) The flow was set at 1 ml/min to wash absorbance compartment. 4) After washing, the column loaded by drop-by-drop technique. 5) The computer has been set up at pressure 0.3 MPa. 6) The injection valve was clean with water and then used buffer. 7) 3 ml buffer was loaded into injection valve. 8) The fraction collector was set up into position 1 by manually. 9) The wizard icon affinity was selected then column brand was clicked. 10) The UV selected at 280 nm 11) The empty loop 100p was set up at 3 ml. 12) The set flow through fractionation was set up at 1ml/min. 13) The elution fractionation was set up at 1ml/min. 14) The target concentration was set up at 100%. 15) The gradient length was set up at 20-coloumn volume. 16) The data of step 8-15 was saved to a file. 17) If bugs exist for flow through, the block flow through fractionation was set up to overcome them. 18) After 15 minutes, the bound and unbound protein that form peaks in the graph was check up.