Editing 1234 1810M1wt1-165 purification (section)

== Protein Purification<br>  ==

2014/12/12-2014/12/19<br> SHANYI 

1234 pET30b-M1wt-1-165 <br> 

culrured on 12/13/2014<br> 

Tuner(DE3)plysN ( transformed on Dec.12), culture overnight&nbsp; 0.2mM IPTG RT induce 2hour, cell lysed<br> 

lysis buffer PH is 8.0, containing 10mM Imidazole<br> 

[[Image:2379 1352 1234purification.jpg]]<br> 

lane5-12: 1234 M1wt1-165 eluted from Ni column (elution buffer:25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,)<br> 

combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff<br> 

connect to FPLC, wash column with 20mM Tris-Hcl PH8.0 50mM NaCL, then elute with 50-500ml Nacl PH8.0, finally use 1M Nacl to elute protein 

[[Image:2379 1234 1810purification.jpg]] 

lane1: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 25 ==&gt;250mM Nacl<br> 

lane2: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 46==&gt;450mM Nacl 

lane3: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 49==&gt;1M Nacl<br> 

lane4: 1234 pET30b-M1wt-1-165, culrured on 12/15/2014, lysate supernatant(Tuner(DE3)plysN , 0.4mM IPTG RT induce O/N, 15.5g/4L)<br> 

lane5: 1810 pT7A30-M1wt-1-165, culrured on 12/15/2014, lysate supernatant(Tuner(DE3)plysN , 0.2mM IPTG RT induce O/N, 15.5g/4L)<br> 

lane6: marker<br> 

load 1234,1810 lysate to Ni column,

wash with bufferA, then wash with 35mM Imidazole, elute with elution buffer(25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,)<br>

[[Image:2379_1234_1810purification2.jpg]]<br>

lane6: Ni column 10mM Imidazole wash flowthrough<br>

lane7-8:Ni column 35mM Imidazole wash flowthrough<br>

lane9-12: protein eluted from Ni column<br>

combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff

connect to FPLC, wash column with 20mM NaPO4 PH7.0, 50mM Nacl, then elute with 50-500ml Nacl PH8.0, finally use 1M Nacl to elute protein <br>

[[Image:2379_1234_1810purification3.jpg]]

lane1-3: protein eluted from spff( 12/16/2014, f43,f45,f48)

lane4-8: protein eluted from spff(12/18/2014,f9,f11,f29,f31,f44)

dissolve pellet in 4M urea PH8.0 buffer,load to Ni column

lane10: dissolved protein

lane11: Ni flowthrough

[[Category:Protein_Purification]]