Paul.xu at 22:46, 31 January 2011

2011-01-31T22:46:51Z

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MBP-VP16 purification

 

N-6XHis--MBP--TEVsite--VP16--C-6XHis

1, load (bacterial lysate) supernatant to Ni columns

2, wash with washing buffer and then  elute with buffer B

3, load to MBP column, wash with PBS, elute protein with 10 mM maltose in PBS

4, digest eluate of step 3 O/N with 20 ul 1 mg/ml TEV

5, load digested protein sol into Ni beads column

6, wash with washing buffer

7, elute with buffer B

8, load eluate from step 7 into MBP column, collect flow thru.

9, wash with PBS

10, elute with 10mM maltose in PBS

MW      1     2      3      4      5      6      7       8       9

[[Image:Vp16 MBP purification.jpg]]

 

MW: 250, 130, 100, 70 (red), 55, 35, 25 (red), 15, 10 

MBP: 42 kDa 

VP16: 55.6 kDa

1, Sample from step 3 (undigested)

2, Sample from step 4 (TEV digested)

3, Sample from step 7 (TEV digested)

4, Sample from step 10 (MBP column eluate)

5, sample from step 8 (MBP column FT)

6, sample from step 6 (wash)

7, Sample from step 9 (wash)

8, Sample from step 3 (undigested), another lot.

9, unknown sample. --[[User:Paul.xu|Paul.xu]] 18:44, 28 January 2011 (EST)

 

 

[[Image:Mbp_vp16_purification.jpg]] 

1,  sample from step 8 (MBP column FT) 

2, sample from step 8 (MBP column FT) 

3, sample from step 3 (undigested) 

4, sample from step 4 (TEV digisted)

5, MBP-VP16 after MBP column

6, VP16 NusA after digestion Ni eluate

7 VP16-GST

8, Vp16-His

10, VP16-NusA after digestion flow through

--[[User:Paul.xu|Paul.xu]] 17:40, 31 January 2011 (EST)

 

[[Category:Protein_Expression]]

VP16-expression 2 - Revision history

Paul.xu at 22:46, 31 January 2011