Paul.xu at 19:30, 12 December 2010

2010-12-12T19:30:02Z

New page

[[Image:Aldolase Vp16 MBP fusion expression.jpg|400x400px|Aldolase Vp16 MBP fusion expression.jpg]]

Expression of MBP-Aldolase and MBP-VP16 in BL21 (DE3) 

MW: 10(bottom), 17, 28(red), 36, 55, 72(red), 95, 130, 250. 

1, His-SM2 

2, pDB.His.MBP-Aldolase, after purification on Ni and  Maltose columns 

3, FT, flow through, Maltose column 

4, pDB.His.MBP-VP16, uninduced BL21(DE3),  eluate from Ni beads. 

5, pDB.His.MBP-VP16, IPTG induced BL21(DE3), clone 1, eluate from Ni beads. 

6, pDB.His.MBP-VP16, IPTG induced BL21(DE3), clone 2, eluate from Ni beads. 

7, pET28b-VP16, uninduced BL21(DE3), eluate from Ni beads. 

8, pET28b-VP16, IPTG induced BL21(DE3), clone 1, eluate from Ni beads. 

9, pET28b-VP16, IPTG induced BL21(DE3), clone 2, eluate from Ni beads. 

 

[[Image:Vp16-MBP expression.jpg]] 

Expression of MBP-Aldolase and MBP-VP16 in BL21 (DE3)

MW: 10(bottom), 17, 28(red), 36, 55, 72(red), 95, 130, 2 

1, pDB.His.MBP-Aldolase after Ni and Maltose columns 

2, pDB.His.MBP-VP16, flow through after Ni and Maltose columns 

3, pDB.His.MBP-Vp16, after Ni and maltose columns 

4, pDB.His.MBP-Vp16, after Ni and maltose columns 

5, Superfolder GFP

6, Superfolder GFP-TPA fusion (forgot to add protein sample).

7, G-Luciferase

8, Insuin

9, Infared Fluorescent protein.

--[[User:Paul.xu|Paul.xu]] 15:42, 5 November 2010 (EST)

[[Image:Vp16 NusA MBP GST expression.jpg]] 

Expression of VP16 fusions. 

1, GST-VP16 (pDB.His.GST-VP16), lysate supernatant, Ni beads pulldown 

2, GST-VP16 (pDB.His.GST-VP16), pellet from lysis, dissolve in 8M Urea, Ni beads pulldown 

3, GST-VP16 (pDB.His.GST-VP16), lysate from 1 L culture, after Ni column purification 4, MBP-VP16 (pDB.His.MBP-VP16), lysate supernatant, Ni beads pulldown 5, MBP-VP16 (pDB.His.MBP-VP16), pellet from lysis, dissolve in 8M Urea, Ni beads pulldown 6, MBP-VP16 (pDB.His.MBP-VP16), lysate from 1 L culture, after Ni column purification 

7, NusA-VP16 (pDB.His.NusA-VP16), lysate supernatant, Ni beads pulldown 8, NusA-VP16 (pDB.His.NusA-VP16), pellet from lysis, dissolve in 8M Urea, Ni beads pulldown 9, NusA-VP16 (pDB.His.NusA-VP16), lysate from 1 L culture, after Ni column purification 

MW ladder is: 10 (not shown), 17, 28 (red), 36, 55, 72 (red), 95, 130, 250 

MBP MW: 43 

GST MW: 26 

NusA MW: 57.6 

VP16: 52.4 kDa, MBP-VP16: 95; GST-Vp16: 78; NusA-Vp16: 110 kDa 

--Paul.xu 12:06, 13 November 2010 (EST) 

 

[[Image:NusA VP16 TEV Digestion 2.jpg]] 

20 ml of dialysed Vp16-NusA in PBS digested with 50 ul TEV o/n at room temp, ~22 oC. 

take 10 ul to run on SDS-PAGE gel. 

Lane 1 is MW marker, 10, 17, 28 (red), 36, 55, 72 (red), 95, 130, 250. Lane 2, undigested partial purified VP16-NusA lane 3, after digestion, mixture of VP16 and NusA, both of them are about 53 kDa. But they run larger than 55 kDa. 

 

[[Image:NusA VP16 TEV Digestion.jpg]] 

1, MW marker, 10, 17, 28 (red), 36, 55, 72 (red), 95, 130, 250. 

2, VP16-NusA, undigested 

3, VP-16-NusA, digested 

4, TEV 

5, old digested VP16-NusA, 10 ul + 1 ul TEV 

6, old digested VP16-NusA, 15 ul + 1 ul TEV 

7, old digested VP16-NusA, 20 ul + 1 ul TEV 

8, BFP, 10 ul 

9, sfGFP-TPA 

10, sfGFP-TPA 

11, VP16-NusA, undigested

12, VP-16-NusA, digested 

 --[[User:Paul.xu|Paul.xu]] 18:47, 19 November 2010 (EST)

[[Image:VP16_purification_WB.jpg]]

MW 10, 17, 28 (Red), 36, 55, 72(Red), 95, 130, 250

1, Vp16-6XHis

2, VP16NusA Eluate (eluated with buffer B )

3, MBP-Vp16 after MBP beads

4, MBP-VP16 after Ni column

5, VP16-NusA after digestion, Ni flow through

A: Anti-His WB

B: SDS-PAGE
--[[User:Paul.xu|Paul.xu]] 12:40, 12 December 2010 (EST)

[[Category:Protein_Expression]]

VP16-expression - Revision history

Paul.xu at 19:30, 12 December 2010