https://eln.excellgen.com/index.php?action=history&feed=atom&title=PET28b-Cre-purificationPET28b-Cre-purification - Revision history2026-10-18T06:20:34ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=PET28b-Cre-purification&diff=2906&oldid=prevRuili.su at 17:30, 30 December 20112011-12-30T17:30:29Z<p></p>
<p><b>New page</b></p><div>[[Image:PET28b-KOD & Cre 375--12-14-2011.jpg|317x225px|PET28b-KOD & Cre 375--12-14-2011.jpg]]<sub></sub><br> <br />
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Date:12/09/2011---12/15/2011<br> <br />
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Notebook: Ruili Su<br> <br />
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Page:002869--002871<br> <br />
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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> <br />
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Line1: Supernatant after ultracentrifuge;<br> <br />
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Line2: Flow through from Ni column;<br> <br />
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Line 3: Washed with I<sub>20</sub>(20mM Imidazole) from Ni column; <br> <br />
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Line 5: washed with I<sub>50&nbsp;</sub>from Ni column;<br> <br />
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Line 6: eluted fraction 1 with Buffer B ( containing I<sub>300</sub>) from Ni column;<br> <br />
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Line 7: eluted fraction 2.<br> <br />
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Line 4, 8 through 11 is for PET28b-Cre protein (DNA clone is 375).<br> <br />
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Line 8: Flow through from Ni column; <br> <br />
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Line 9: Washed with I<sub>20</sub> from Ni column;<br> <br />
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Line 10: Washed fraction 1 with I<sub>50</sub> from Ni column;<br> <br />
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Line 11: washed fraction 2 with I<sub>50</sub><sub></sub><sub></sub> from Ni column;<br> <br />
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Line 4: washed fraction 3 with I<sub>50 </sub>from Ni column;<br> <br />
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'''Result'''<br> <br />
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1.<br> <br />
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2. the protein expression for PET28b-Cre is not so good.<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> <br />
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Date: 12/16/2011--12/17/2011<br> <br />
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Notebook: ruili su<br> <br />
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Page: 002872<br> <br />
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Line 1: Protein Cre(PET28b-Cre)&nbsp; flow through from FPLC; (10ul)<br> <br />
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Line 2--5: Protein Cre washed with&nbsp; I<sub>30 </sub>(30 mM imidazole) for fraction 17--20; ()10ul <br />
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Line 6-11 Eluted protein Taq-Muts with buffer B for fractions. (10ul)<sub></sub> <br />
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'''Result''' <br />
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Cre protein had good expression, and the size is correct (~40KD). Futherly, need to dylize with buffer C and buffer D; <br />
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[[Image:PET28b-Cre-12-21-2011.jpg|305x236px|PET28b-Cre-12-21-2011.jpg]] <br />
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Date: 12/19/2011--12/21/2011 <br />
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NotebooK: Ruili Su <br />
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Page: 002873--002874 <br />
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After FPLC, detect protein with Braford Reagent and run SDS-PAGE; <br />
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Line 1--3: Cre washed with I<sub>30</sub> from FPLC; <br />
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Line 4--10: Cre is washed with I<sub>50 </sub>from FPLC. <br />
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'''Result''' <br />
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The protein has good expression level, and combining the fractions to dialyse. <br />
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--[[User:Ruili.su|Ruili.su]] 18:02, 21 December 2011 (EST) <br />
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[[Image:PET28b-Cre-12-29-2011.jpg|310x248px]]<br />
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[[Category:Protein_Purification]]</div>Ruili.su