https://eln.excellgen.com/index.php?action=history&feed=atom&title=1234_1810M1wt1-165_purification 2026-10-18T02:19:29Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1234_1810M1wt1-165_purification&diff=13742&oldid=prev 2014-12-19T19:57:33Z

<p></p> <p><b>New page</b></p><div>== Protein Purification&lt;br&gt; ==<br /> <br /> 2014/12/12-2014/12/19&lt;br&gt; SHANYI <br /> <br /> 1234 pET30b-M1wt-1-165 &lt;br&gt; <br /> <br /> culrured on 12/13/2014&lt;br&gt; <br /> <br /> Tuner(DE3)plysN ( transformed on Dec.12), culture overnight&amp;nbsp; 0.2mM IPTG RT induce 2hour, cell lysed&lt;br&gt; <br /> <br /> lysis buffer PH is 8.0, containing 10mM Imidazole&lt;br&gt; <br /> <br /> [[Image:2379 1352 1234purification.jpg]]&lt;br&gt; <br /> <br /> lane5-12: 1234 M1wt1-165 eluted from Ni column (elution buffer:25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,)&lt;br&gt; <br /> <br /> combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff&lt;br&gt; <br /> <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 <br /> <br /> [[Image:2379 1234 1810purification.jpg]] <br /> <br /> lane1: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 25 ==&amp;gt;250mM Nacl&lt;br&gt; <br /> <br /> lane2: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 46==&amp;gt;450mM Nacl <br /> <br /> lane3: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 49==&amp;gt;1M Nacl&lt;br&gt; <br /> <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)&lt;br&gt; <br /> <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)&lt;br&gt; <br /> <br /> lane6: marker&lt;br&gt; <br /> <br /> load 1234,1810 lysate to Ni column,<br /> <br /> wash with bufferA, then wash with 35mM Imidazole, elute with elution buffer(25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,)&lt;br&gt;<br /> <br /> [[Image:2379_1234_1810purification2.jpg]]&lt;br&gt;<br /> <br /> lane6: Ni column 10mM Imidazole wash flowthrough&lt;br&gt;<br /> <br /> lane7-8:Ni column 35mM Imidazole wash flowthrough&lt;br&gt;<br /> <br /> lane9-12: protein eluted from Ni column&lt;br&gt;<br /> <br /> combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff<br /> <br /> 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 &lt;br&gt;<br /> <br /> [[Image:2379_1234_1810purification3.jpg]]<br /> <br /> lane1-3: protein eluted from spff( 12/16/2014, f43,f45,f48)<br /> <br /> lane4-8: protein eluted from spff(12/18/2014,f9,f11,f29,f31,f44)<br /> <br /> dissolve pellet in 4M urea PH8.0 buffer,load to Ni column<br /> <br /> lane10: dissolved protein<br /> <br /> lane11: Ni flowthrough<br /> <br /> [[Category:Protein_Purification]]</div>

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