Editing 1708 cre purification2014 11 4-6 (section)

== Protein Purification  ==

2014/11/3-2014/11/6<br> SHANYI 

1708 Nico21(DE3)plysN <br> 

purifying using 20ml Ni column (new Ni beads packed in XK 16/20 Column),lysis buffer and buffer A PH is 8.0<br> 

after loading sample. the column turns white 

wash with 10cv Buffer A(5 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole,1 mM 2-Mercaptoethanol) ,<br> 

connect column to FPLC,wash with 35mM Imidazole until OD280 drops to baseline<br> 

then run linear gradient program(10mM to 250 Imidazole in 25 mM NaPO4, pH 7.0 ) on FPLC to elute protein, no peaks<br>FPLC result: method run11/04/2014,10:43:14PM 

Even though where is no peak, chose some fractions to run a gel<br> 

[[Image:2367lysate 2369 2375 1708purification.jpg]]<br> 

lane5:PageRuler Plus protein ladder <br> 

lane6: 1708 expression screening ( culture with black medium)<br> 

lane7: 1708 expression screening( culture with LB)<br> 

lane8: lysate(black medium)<br> 

lane9: Ni column flowthrough<br> 

lane10: Ni column wash flowthrough(35mM Imidazole)<br> 

lane11-12: protein eluted from Ni column (Imidazole concentration &gt;150mM) PH7.0 <br> 

MOST OF THE PROTEINS AGGREGATED ON THE COLUMN.<br> 

combine fractions, load to 20ml sp FF (contain 250mM Nacl,PH7.0 )<br> 

wash with 200ml 10cv SP Washing Buffer(50 mM Phosphate Buffer, pH 7.0, 500 mM NaCl, 0.1 mM EDTA.&nbsp; 1 mM 2-Mercaptoethanol ) <br> 

Elute with SP Elution Buffer( 50 mM Tris-HCl, pH 8.0, 1.5 M NaCl, 5% glycerol, 0.1 mM EDTA.&nbsp; 1 mM&nbsp; 2-Mercaptoethanol ) <br> 

Run gel<br> 

[[Image:1708 eluted from sp.jpg]]<br> 

11/04<br> 

culture another 4L cre, when OD600&gt;1.5 use 0.2 mM IPTG RT induce o/n 16g/4L<br> 

lyse cell using Lysis Buffer: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM beta-ME,<br> 

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

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

&nbsp;Elute protein with 150 ml or more of Ni Elution Buffer (50 mM Phosphate Buffer, pH 7.0, 250 mM NaCl, 100 mM arginine, 300 mM imidazole, 10% glycerol) directly to 20 ml SP FF, preequilibrated with SP Dilution Buffer.&nbsp; 

Wash SP FF with 10 CV of SP Washing Buffer until OD280 reaches baseline, <br> 

Elute with 5~10 CV of SP Elution Buffer, collect 10 ml fractions 

[[Image:2367 1708purification11 05 2014.jpg]]<br> 

lane5: lysate<br> 

lane6: flowthrough from Ni column<br> 

lan7: lysate purified with 75ul Ni beads<br> 

[[Image:1708cre eluted from sp-2367eluted from Ni 2014 11 06.jpg]]<br>lane1-4: cre eluted from sp ff<br> 

combine&nbsp; to&nbsp; dialyse<br> 

2.93mg/ml 40ml before dialysis, 6.7mg/ml 18ml after dialysis<br> 

lot number:75-1708 

[[Category:Protein_Purification]]