Cre1708 expression screening 2367 2368 2014 10 29

Protein Expression

2014/10/29
SHANYI

Lane	Name	Protein Name	Clone ID	Host	Induction	Purification	pI/Mw	express
1	pT7A3-HTNCre		1708	1L Nico21(DE3)plysN	0.5 mM IPTG  37oC 4.75h	Ni beads
2					0.5 mM IPTG 37oC 4.75h	Ni beads		wash with 35mM Imidazole
3				1L Nico21(DE3)plysN	0.5 mM IPTG 30oC 4.75h	Ni beads
4					0.5 mM IPTG 30oC 4.75h	Ni beads		wash with 35mM Imidazole
5				1L Nico21(DE3)plysN	0.5 mM IPTG(ours) RT O/N	Ni beads
6				1L Nico21(DE3)plysN	0.5 mM IPTG RT O/N	Ni beads
7	PageRuler Plus protein ladder
8	pCold-MBP-(Ent-TEV)-HCMVUL98		2367	15 ml, Nico21(DE3)plysN	0.2 mM IPTG RT O/N	Ni beads
9	pCold-MBP-(Ent-TEV)-HCMVUL98		2367	15 ml, Tuner(DE3)plysN-tev	0.2 mM IPTG RT O/N	Ni beads
10	pT7A3-MBP-(TEV)-HCMVUL98		2368	15 ml, Nico21(DE3)plysN	0.2 mM IPTG RT O/N	Ni beads	110
11	pT7A3-MBP-(TEV)-HCMVUL98		2368	15 ml, Tuner(DE3)plysN-tev	0.2 mM IPTG RT O/N	Ni beads	67+42
12	pCold-MBP-(Ent-TEV)-HCMVUL98		2367	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT O/N	Ni beads		pellet from lane8

2014/10/30

combine cre lysate from 4L 1708 Nico21(DE3)plysN

purifying using 20ml Ni column (new Ni beads packed in XK 16/20 Column),lysisbuffer and buffer A are NaPO4 buffer PH7.4

after loading sample. the column turns white

wash with 5cv 5mM Imidazole , then elute, when the peak comes out, stop FPLC at Imidazole concentration is 150mM

FPLC result: method run10/31/2014,5:08:20AM

lane1:600ul lysate (purified with 75ul screening Ni beads )

lane2:Ni column flowthrough(purified with 75ul screening Ni beads )

lane3-7:protein eluted from Ni column

lane8:PageRuler Plus protein ladder

lane9: protein eluted from Ni column combined together 30ul

lane10: sp ff flowthrough 30ul

lane11: protein eluted from sp ff(250-1M NaCl)

lane12: protein eluted from sp ff(2M NaCl 100mM Arginine 20mM Tris-Hcl)