Excellgen at 21:14, 26 November 2014

2014-11-26T21:14:09Z

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== Protein Purification ==

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, 

[[Image:2367lysate 2369 2375 1708purification.jpg]]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)

[[Image:1708cre eluted from sp-2367eluted from Ni 2014 11 06.jpg]]

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

[[Image:2367 ni giga650q 11 09.jpg]]

lane1-7: protein eluted from Giga Q650

not pure enough, combine, then load to 5ml Histrap HP ---->20ml QHP(50mM-1M Nacl elute 300ml)

[[Image:2367 ni giga650q ni qhp2014 11 09.jpg]]fraction20-31

combine fraction 22-28, dilute,load to 20ml Q HP again, eluted with 50-500mM Nacl 300ml

[[Image:2367 ni giga650q ni qhp qhp2014 11 09 cre2014 11 11.jpg]] 

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 

[[Image:2367_2115.jpg]] 

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

 

 

 

 

[[Category:Protein_Purification]]

2367purification pCold-MBP-(Ent-TEV)-HCMVUL98 - Revision history

Excellgen at 21:14, 26 November 2014