Editing 1352cre 1810M1wt1-165purification 2014 12

== Protein purification  ==

2014/12/10-2014/12/13<br> SHANYI 

1352&nbsp;Cre (without Tat and NLS)&nbsp;&nbsp; lot number:79-1352<br>

Nico21(DE3)plysN&nbsp; 0.5mM IPTG RT induce overnight&nbsp; 8L 42.7g<br> 

purifying using 20ml Ni column (two),lysis buffer and buffer A PH is 8.0<br>

wash with 10cv Buffer A(5 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole,1 mM 2-Mercaptoethanol) ,until OD280 drops to baseline. 

&nbsp;Elute protein with 150 ml cre Ni Elution Buffer (50 mM Phosphate Buffer, pH 7.0, 250 mM NaCl, 100 mM arginine, 300 mM imidazole, 10% glycerol) directly to 5ml QFF+20 ml SP FF, preequilibrated with SP Dilution Buffer.&nbsp; 

Wash SP FF with&nbsp; SP Washing Buffer until OD280 reaches baseline, <br> 

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

[[Image:1352_1810stock_purification.jpg]]<br> 

lane1: 1352 lysate<br>

lane2: 1352 Ni column flowthrough<br>

lane3: 1352 Ni column wash flowthrough<br>

lane4: 1352 sp loading flowthrogh<br>

lane5: 1352 sp wash flowthrough<br>

lane6: 1352 protein eluted from sp column<br>

lane7: PageRuler Plus Prestained protein ladder <br>

lane8: 1810 protein eluted from Ni column, diluted <br>

lane9: 1810 protein eluted from Ni column<br>

lane10:1810 Ni column wash flowthrough<br>

lane11:1810 Ni column loading flowthrough<br>

lane12:1810 lysate<br>

1352 cre without Tat and NLS cannot bind to sp column when NaCl concentration is 250mM, need to dilute with 5CV cre dilution buffer first, then load to sp<br>

1810 protein size is not right, the stock in -80oC&nbsp; maybe not right.

[[Image:1352_purification.jpg]]1352 CRE eluted from sp column

<br>[[Category:Protein_Purification]]