Vp16-purification-2
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
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)
--Paul.xu 17:09, 28 April 2011 (EST)
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)
--Ruili.su 15:28, 29 April 2011 (EST)
Page: 002744
Date: 05/03/2011
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.
--Ruili.su 16:24, 4 May 2011 (EST)