Editing 2379cre-flppurification2014 12

== Protein Purification  ==

2014/12/12-2014/12/19<br> SHANYI 

'''2379 pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H'''<br> 

Nico21(DE3)plysN&nbsp; 0.2mM IPTG RT induce overnight&nbsp; 8L 34.2g<br> 

&nbsp;lysis buffer&nbsp; PH is 8.0, containing 10mM Imidazole<br> 

[[Image:2379 1352 1234purification.jpg]]<br> 

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

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

load 2379 lysate supernatant to 20ml Ni column <br> 

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) <br> 

[[Image:2379 1234 1810purification.jpg]]<br> 

lane8-12: cre-flp eluted from Ni column<br> 

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

wash with 50 mM Phosphate Buffer, pH 7.0, 50 mM NaCl<br> 

elute with cre elution buffer containing 5%glycerol 

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

lane1-4: cre-flp eluted from sp FF<br> 

concentrate, use 1:50 TEV digest protein. 

[[Image:2379_1234_1810purification3.jpg]]

lane12: concentrated cre-flp.<br> 

<br> 

[[Category:Protein_Purification]]