1951 1952

Protein Expression

2014/1/14
Paul

Lane	Name	Protein Name	Clone ID	Host	Induction	Purification	pI/Mw
1	pT7A3-HC-yFLP	Tat-yFLP	1951	5 ml, NEB Turbo	1 mM IPTG  RT o/n	Ni beads	9.76 / 52.8
2	pT7A3-HC-yFLP	Tat-yFLP	1951	5 ml, NEB Turbo	1 mM IPTG RT o/n	Ni beads	9.76 / 52.8
3	pT7A3-HC-yFLP	Tat-yFLP	1951	5 ml, NEB Turbo	1 mM IPTG RT o/n	Ni beads	9.76 / 52.8
4	pT7A3-Tat-Flpo	Tat-Flpo	1952	5 ml, NEB Turbo	1 mM IPTG RT o/n	Ni  beads	9.76 / 52.8
6	pT7A3-Tat-Flpo	Tat-Flpo	1952	5 ml, NEB Turbo	1 mM IPTG RT o/n	Ni beads	9.76 / 52.8
7	pT7A3-HC-yFLP	Tat-yFLP	1951	5 ml, Nico21(DE3)pLysN	1 mM IPTG RT o/n	Ni beads	9.76 / 52.8
8	pT7A3-HC-yFLP	Tat-yFLP	1951	5 ml, Nico21(DE3)pLysN	1 mM IPTG RT o/n	Ni beads	9.76 / 52.8
9	pT7A3-HC-yFLP	Tat-yFLP	1951	5 ml, Nico21(DE3)pLysN	1 mM IPTG RT o/n	Ni beads	9.76 / 52.8
10	pT7A3-Tat-Flpo	Tat-Flpo	1952	5 ml, Nico21(DE3)pLysN	1 mM IPTG RT o/n	Ni beads	9.76 / 52.8
11	Fermentas PageRuler Plus
12	purified T4 Ligase 2 ul of 38 mg/ml

Tat-yFLP has better expression than Tat-Flpo

Purification of Tat-yFLP on Ni column.

Lysate from 8 L culture, Nico21(DE3)pLysN, 0.2 mM IPTG o/n. Load to 15 ml Ni column,

Lane 1 first Ni column eluate and concentrated.

Lane 2 and 4~12: Load 5X diluted protein solution to 2nd 5 ml GE pharmacia Ni column, elute by linear gradient of Imidazole. Fractions containing proteins

Looks like protein expression is low, also not pure (lot of degradation)