Excellgen at 19:45, 19 December 2014

2014-12-19T19:45:46Z

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== 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 

[[Image:2379 1352 1234purification.jpg]] 

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) 

[[Image:2379 1234 1810purification.jpg]] 

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

[[Image:2379 1234 1810purification2.jpg]]

lane1-4: cre-flp eluted from sp FF 

concentrate, use 1:50 TEV digest protein.

[[Image:2379_1234_1810purification3.jpg]]

lane12: concentrated cre-flp. 

 

[[Category:Protein_Purification]]

2379cre-flppurification2014 12 - Revision history

Excellgen at 19:45, 19 December 2014