VP16-expression

Error creating thumbnail: File with dimensions greater than 12.5 MP

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.

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.

--Paul.xu 15:42, 5 November 2010 (EST)

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)

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.

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

--Paul.xu 18:47, 19 November 2010 (EST)

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 --Paul.xu 12:40, 12 December 2010 (EST)