1708 cre purification2014 11 4-6

Protein Purification

2014/11/3-2014/11/6
SHANYI

1708 Nico21(DE3)plysN

purifying using 20ml Ni column (new Ni beads packed in XK 16/20 Column),lysis buffer and buffer A PH is 8.0

after loading sample. the column turns white

wash with 10cv Buffer A(5 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole,1 mM 2-Mercaptoethanol) ,

connect column to FPLC,wash with 35mM Imidazole until OD280 drops to baseline

then run linear gradient program(10mM to 250 Imidazole in 25 mM NaPO4, pH 7.0 ) on FPLC to elute protein, no peaks
FPLC result: method run11/04/2014,10:43:14PM

Even though where is no peak, chose some fractions to run a gel

lane5:PageRuler Plus protein ladder

lane6: 1708 expression screening ( culture with black medium)

lane7: 1708 expression screening( culture with LB)

lane8: lysate(black medium)

lane9: Ni column flowthrough

lane10: Ni column wash flowthrough(35mM Imidazole)

lane11-12: protein eluted from Ni column (Imidazole concentration >150mM) PH7.0

MOST OF THE PROTEINS AGGREGATED ON THE COLUMN.

combine fractions, load to 20ml sp FF (contain 250mM Nacl,PH7.0 )

wash with 200ml 10cv SP Washing Buffer(50 mM Phosphate Buffer, pH 7.0, 500 mM NaCl, 0.1 mM EDTA.  1 mM 2-Mercaptoethanol )

Elute with SP Elution Buffer( 50 mM Tris-HCl, pH 8.0, 1.5 M NaCl, 5% glycerol, 0.1 mM EDTA.  1 mM  2-Mercaptoethanol )

Run gel

11/04

culture another 4L cre, when OD600>1.5 use 0.2 mM IPTG RT induce o/n 16g/4L

lyse cell using Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM beta-ME,

load to 20ml Ni column( old Ni beads simply packed in yellow column)

Wash column with 300 ml Buffer A until OD280 drops to baseline.

 Elute protein with 150 ml or more of Ni Elution Buffer (50 mM Phosphate Buffer, pH 7.0, 250 mM NaCl, 100 mM arginine, 300 mM imidazole, 10% glycerol) directly to 20 ml SP FF, preequilibrated with SP Dilution Buffer. 

Wash SP FF with 10 CV of SP Washing Buffer until OD280 reaches baseline,

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

lane5: lysate

lane6: flowthrough from Ni column

lan7: lysate purified with 75ul Ni beads

lane1-4: cre eluted from sp ff

combine  to  dialyse

2.93mg/ml 40ml before dialysis, 6.7mg/ml 18ml after dialysis

lot number:75-1708