Editing PET28b-Cre-purification

[[Image:PET28b-KOD & Cre 375--12-14-2011.jpg|317x225px|PET28b-KOD & Cre 375--12-14-2011.jpg]]<sub></sub><br> 

Date:12/09/2011---12/15/2011<br> 

Notebook: Ruili Su<br> 

Page:002869--002871<br> 

Line 1 through 7 is for PET28b-KOD protein (DNA clone is 480)--except line 4 (Transform with Rosetta BL21 (DE3) PlysS and IPTG induced); <br> 

Line1: Supernatant after ultracentrifuge;<br> 

Line2: Flow through from Ni column;<br> 

Line 3: Washed with I<sub>20</sub>(20mM Imidazole) from Ni column; <br> 

Line 5: washed with I<sub>50&nbsp;</sub>from Ni column;<br> 

Line 6: eluted fraction 1 with Buffer B ( containing I<sub>300</sub>) from Ni column;<br> 

Line 7: eluted fraction 2.<br> 

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Line 4, 8 through 11 is for PET28b-Cre protein (DNA clone is 375).<br> 

Line 8: Flow through from Ni column; <br> 

Line 9: Washed with I<sub>20</sub> from Ni column;<br> 

Line 10: Washed fraction 1 with I<sub>50</sub> from Ni column;<br> 

Line 11: washed fraction 2 with I<sub>50</sub><sub></sub><sub></sub> from Ni column;<br> 

Line 4: washed fraction 3 with I<sub>50 </sub>from Ni column;<br> 

'''Result'''<br> 

1.<br> 

2. the protein expression for PET28b-Cre is not so good.<br> 

<br> 

<br> 

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[[Image:PET28b-Cre & Taq Muts--12-16-2011.jpg|311x219px|PET28b-Cre & Taq Muts--12-16-2011.jpg]]<br> 

Date: 12/16/2011--12/17/2011<br> 

Notebook: ruili su<br> 

Page: 002872<br> 

Line 1: Protein Cre(PET28b-Cre)&nbsp; flow through from FPLC; (10ul)<br> 

Line 2--5: Protein Cre washed with&nbsp; I<sub>30 </sub>(30 mM imidazole) for fraction 17--20; ()10ul 

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Line 6-11 Eluted protein Taq-Muts with buffer B for fractions. (10ul)<sub></sub> 

'''Result''' 

Cre protein had good expression, and the size is correct (~40KD). Futherly, need to dylize with buffer C and buffer D; 

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[[Image:PET28b-Cre-12-21-2011.jpg|305x236px|PET28b-Cre-12-21-2011.jpg]] 

Date: 12/19/2011--12/21/2011 

NotebooK: Ruili Su 

Page: 002873--002874 

After FPLC, detect protein with Braford Reagent and run SDS-PAGE; 

Line 1--3: Cre washed with I<sub>30</sub> from FPLC; 

Line 4--10: Cre is washed with I<sub>50 </sub>from FPLC. 

'''Result''' 

The protein has good expression level, and combining the fractions to dialyse. 

--[[User:Ruili.su|Ruili.su]] 18:02, 21 December 2011 (EST) 

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[[Image:PET28b-Cre-12-29-2011.jpg|310x248px]]







[[Category:Protein_Purification]]