Editing Vp16-purification-2

Notebook: Ruili #<br> 

Page: 002735<br> 

Date: 04/28/2011<br> 

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

<br> 

Protein was partially purified by Ni column. 

Digest Ni-bead purifiied VP16-MBP with TEV&nbsp; 

2nd round purification with Amyloase beads. <br> 

<br> 

'''TEV Digestion method: '''<br> 

5 mM DTT<br> 

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 

<br> 

'''Chromatography method:'''<br> 

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

2. Wash beads with H2O 

3. Equibriate beads with PBS<br> 

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

5. Collect flow through<br> 

6. Wash beads with PBS, collect wash<br> 

7. Elute bound MBP with 10 mM maltose in PBS.<br> 

'''Regenerate amylose beads column:''' 

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

2. Wash with PBS;&nbsp; 

3. Wash with 0.1% SDS; 

4. Wash with H<sub>2</sub>O; 

5. Wash with PBS. 

<br> 

'''SDS-PAGE''' 

<br> 

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

<br> 

[[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&nbsp; 

3. Flow through from amylose column 

4. Wash with PBS 

5. Eluate with 10 mM Maltose in PBS<br> 

V: Vp16 

M: Maltose-Binding Protein 

F: fusion protein (uncut)&nbsp; 

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

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

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

2. Flow thorugh from amylose column;<br> 

3. Washed with PBS;<br> 

4. Elute with 10mM Maltose in PBS; <br> 

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

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

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

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

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

Page: 002744 

Date: 05/03/2011<br> 

[[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. 

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

[[Category:Protein_Purification]]