PET28b-Cre-purification

Date:12/09/2011---12/15/2011

Notebook: Ruili Su

Page:002869--002871

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);

Line1: Supernatant after ultracentrifuge;

Line2: Flow through from Ni column;

Line 3: Washed with I₂₀(20mM Imidazole) from Ni column;

Line 5: washed with I₅₀ from Ni column;

Line 6: eluted fraction 1 with Buffer B ( containing I₃₀₀) from Ni column;

Line 7: eluted fraction 2.

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

Line 8: Flow through from Ni column;

Line 9: Washed with I₂₀ from Ni column;

Line 10: Washed fraction 1 with I₅₀ from Ni column;

Line 11: washed fraction 2 with I₅₀ from Ni column;

Line 4: washed fraction 3 with I₅₀ from Ni column;

Result

1.

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

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Date: 12/16/2011--12/17/2011

Notebook: ruili su

Page: 002872

Line 1: Protein Cre(PET28b-Cre)  flow through from FPLC; (10ul)

Line 2--5: Protein Cre washed with  I₃₀ (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)

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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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 I₃₀ from FPLC;

Line 4--10: Cre is washed with I₅₀ from FPLC.

Result

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

--Ruili.su 18:02, 21 December 2011 (EST)

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