Ruili.su at 21:24, 4 May 2011

2011-05-04T21:24:44Z

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Notebook: Ruili # 

Page: 002735 

Date: 04/28/2011 

Title: Purification of VP16 by affnity chromatography on amylose-agarose beads. 

 

Protein was partially purified by Ni column.

Digest Ni-bead purifiied VP16-MBP with TEV 

2nd round purification with Amyloase beads. 

 

'''TEV Digestion method: ''' 

5 mM DTT 

2 mM EDTA

1:50 TEV, cold room O/N

1.5 mg/ml protein, 25 ml VP16-MBP: add 750 ul 1 mg/ml TEV

125 ul 1 M DTT

100 ul 0.5 M EDTA

 

'''Chromatography method:''' 

1. Wash beads with H2O, PBS, H2O, then regenerate with 0.1% SDS (see next step) 

2. Wash beads with H2O

3. Equibriate beads with PBS 

4. Load 25 ml TEV digested VP16-MBP (~1.5 mg/ml) 

5. Collect flow through 

6. Wash beads with PBS, collect wash 

7. Elute bound MBP with 10 mM maltose in PBS. 

'''Regenerate amylose beads column:'''

1. Wash beads with 3--4 volume of beads;

2. Wash with PBS; 

3. Wash with 0.1% SDS;

4. Wash with H2O;

5. Wash with PBS.

 

'''SDS-PAGE'''

 

[[Image:Vp16 purification2 4 28 2011.jpg|253x354px|Vp16 purification2 4 28 2011.jpg]]

 

[[Image:Vp16 purification 4 28 11.jpg|209x333px|Vp16 purification 4 28 11.jpg]]

MW: 10, 15, 25 (red), 35, 56, 70(red), 100, 130, 250

1. Undigested Vp-16-MBP

2. TEV-Digested Vp16-BMP 

3. Flow through from amylose column

4. Wash with PBS

5. Eluate with 10 mM Maltose in PBS 

V: Vp16

M: Maltose-Binding Protein

F: fusion protein (uncut) 

--[[User:Paul.xu|Paul.xu]] 17:09, 28 April 2011 (EST)

[[Image:Vp16-MBP purification 4 29--2011.jpg]] 

1. TEV-Digested Vp16-BMP on 4/28/2011(overnight); 

2. Flow thorugh from amylose column; 

3. Washed with PBS; 

4. Elute with 10mM Maltose in PBS; 

5. Flow thorugh from amylose column (4/28/2011) 

6. Re-flow through (continue to do from NO.5 ) 

7. Re-washed with PBS (fcontinue to do from NO. 6) 

8. Re-elute 10mM Maltose in PBS (continue to do from NO.7) 

9. TEV-Digested Vp16-BMP on 4/27/2011 (overnight) --[[User:Ruili.su|Ruili.su]] 15:28, 29 April 2011 (EST)

Page: 002744

Date: 05/03/2011 

[[Image:Vp16 purification 5 04 11.jpg]]

VP16 purification running blue Gel

1. Combine the protein from tube 26-28;

2. Flow through from combine protein;

3. Wash the combination with PBS;

4. Elute the combination with 10 mM Maltose in PBS

5. Elute the residul of tube 30

6. Wash the residul of the tube 30 with PBS.

 --[[User:Ruili.su|Ruili.su]] 16:24, 4 May 2011 (EST)

[[Category:Protein_Purification]]

Vp16-purification-2 - Revision history

Ruili.su at 21:24, 4 May 2011