Editing VP16-expression 2

MBP-VP16 purification 

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

1, load (bacterial lysate) supernatant to Ni columns 

2, wash with washing buffer and then&nbsp; 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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&nbsp;&nbsp;&nbsp;&nbsp; 2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 6&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 7&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 8&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9 

[[Image:Vp16 MBP purification.jpg]] 

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

MBP: 42 kDa<br> 

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) 

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[[Image:Mbp_vp16_purification.jpg]]<br>

1,&nbsp; sample from step 8 (MBP column FT) <br>

2, sample from step 8 (MBP column FT)<br>

3, sample from step 3 (undigested) <br>

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)

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[[Category:Protein_Expression]]