2367purification pCold-MBP-(Ent-TEV)-HCMVUL98
Protein Purification
[edit]2014/11/4-2014/11/11
2367 pCold-MBP-(Ent-TEV)-HCMVUL98
8L Tuner(DE3)plysN-TEV 0.2mMIPTG RT induce overnight 35.8g
Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 1 mM beta-ME,
lane1: 2367 lysate purified with 75ul Ni beads
load to 20ml Ni column( old Ni beads simply packed in blue column)
Wash column with 300 ml Buffer A until OD280 drops to baseline.
Elute protein with BufferB (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 250 mM imidazole, 10% glycerol)
lane6-12: 2367 protein eluted from Ni column
combine fraction1-7, dilute with 1cv 25 mM Tris-HCl, pH 8.0, load to 5ml Giga Q650
Wash with 10 CV 25 mM Tris-HCl, pH 8.0, 50mM NaCl,until OD280 reaches baseline,
Elute with 50mM-1M Nacl
lane1-7: protein eluted from Giga Q650
not pure enough, combine, then load to 5ml Histrap HP ---->20ml QHP(50mM-1M Nacl elute 300ml)
fraction20-31
combine fraction 22-28, dilute,load to 20ml Q HP again, eluted with 50-500mM Nacl 300ml
lane1-7: protein eluted from QHP
2014/11/13-2014/11/19
2367 pCold-MBP-(Ent-TEV)-HCMVUL98
8L Nico21(DE3)plysN 0.2mMIPTG RT induce overnight 38.7g
Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 1 mM beta-ME,0.1%NP40
load to yellow Ni column
lane1: 2367 lysate
lane2: Ni column flowthrough
lane3: 10mM Imidazole wash flowthrough
lane4: 20mM Imidazole wash flowthrough
lane5: PageRuler Plus protein ladder
lane6-10: protein eluted from Ni column
lane11-15: 2115 flp pellet