1352cre 1810M1wt1-165purification 2014 12

Protein purification

2014/12/10-2014/12/13
SHANYI

1352 Cre (without Tat and NLS)   lot number:79-1352

Nico21(DE3)plysN  0.5mM IPTG RT induce overnight  8L 42.7g

purifying using 20ml Ni column (two),lysis buffer and buffer A PH is 8.0

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 150 ml cre Ni Elution Buffer (50 mM Phosphate Buffer, pH 7.0, 250 mM NaCl, 100 mM arginine, 300 mM imidazole, 10% glycerol) directly to 5ml QFF+20 ml SP FF, preequilibrated with SP Dilution Buffer. 

Wash SP FF with  SP Washing Buffer until OD280 reaches baseline,

Elute with 5~10 CV of cre SP Elution Buffer, collect fractions

lane1: 1352 lysate

lane2: 1352 Ni column flowthrough

lane3: 1352 Ni column wash flowthrough

lane4: 1352 sp loading flowthrogh

lane5: 1352 sp wash flowthrough

lane6: 1352 protein eluted from sp column

lane7: PageRuler Plus Prestained protein ladder

lane8: 1810 protein eluted from Ni column, diluted

lane9: 1810 protein eluted from Ni column

lane10:1810 Ni column wash flowthrough

lane11:1810 Ni column loading flowthrough

lane12:1810 lysate

1352 cre without Tat and NLS cannot bind to sp column when NaCl concentration is 250mM, need to dilute with 5CV cre dilution buffer first, then load to sp

1810 protein size is not right, the stock in -80oC  maybe not right.

1352 CRE eluted from sp column