2379cre-flppurification2014 12

Protein Purification

2014/12/12-2014/12/19
SHANYI

2379 pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H

Nico21(DE3)plysN  0.2mM IPTG RT induce overnight  8L 34.2g

 lysis buffer  PH is 8.0, containing 10mM Imidazole

lane1: 2379 lysate supernatant(use 75ul Ni bead to purify)

lane2: 2379 lysate pellet(use 75ul Ni bead to purify)

load 2379 lysate supernatant to 20ml Ni column

wash with 10cv Buffer A(5 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole,1 mM 2-Mercaptoethanol) ,until OD280 drops to baseline.

Elute protein with cre Ni Elution Buffer (50 mM Phosphate Buffer, pH 7.0, 250 mM NaCl, 100 mM arginine, 300 mM imidazole, 10% glycerol)

lane8-12: cre-flp eluted from Ni column

diluted with 5cv cre dilution buffer, load to 20ml sp FF before loading A280=0.22, after loading A280=0.16

wash with 50 mM Phosphate Buffer, pH 7.0, 50 mM NaCl

elute with cre elution buffer containing 5%glycerol

lane1-4: cre-flp eluted from sp FF

concentrate, use 1:50 TEV digest protein.

lane12: concentrated cre-flp.