Editing 2367purification pCold-MBP-(Ent-TEV)-HCMVUL98

== Protein Purification  ==

2014/11/4-2014/11/11<br> 

2367 pCold-MBP-(Ent-TEV)-HCMVUL98<br> 

8L Tuner(DE3)plysN-TEV 0.2mMIPTG RT induce overnight 35.8g<br> 

&nbsp;Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 1 mM beta-ME,<br> 

[[Image:2367lysate 2369 2375 1708purification.jpg]]lane1: 2367 lysate purified with 75ul Ni beads<br> 

load to 20ml Ni column( old Ni beads simply packed in blue column)<br> 

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

&nbsp;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,&nbsp; dilute with 1cv&nbsp; 25 mM Tris-HCl, pH 8.0, load to 5ml Giga Q650<br> 

Wash&nbsp; with 10 CV &nbsp; 25 mM Tris-HCl, pH 8.0, 50mM NaCl,until OD280 reaches baseline, <br> 

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 ----&gt;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]]<br> 

lane1-7: protein eluted from QHP 

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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<br>

load to yellow Ni column<br>

[[Image:2367_2115.jpg]]<br>

lane1: 2367 lysate<br>

lane2: Ni column flowthrough<br>

lane3: 10mM Imidazole wash flowthrough<br>

lane4: 20mM Imidazole wash flowthrough<br>

lane5: PageRuler Plus protein ladder <br>

lane6-10: protein&nbsp; eluted from Ni column<br>

lane11-15: 2115 flp pellet



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[[Category:Protein_Purification]]