Editing Vp16-purification

[[Image:MBP--vp16 digestion-4-12 .jpg]] 

MBP:42KDa; VP16:55.6Kda;TEV:27 KDa. 

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

<br> 1. NUSA, undigested, from Ni column;<br> 

2. MBP, undigested, from Ni column;<br> 

3. NUSA (1mg/ml ), 1:50 digested by TEV (1mg/ml), from Ni column; 

4. MBP(1 mg/ml), 1:50 digested by TEV (1mg/ml) , from Ni column;<br> 

5. PKA_alpha with pGro7, from Ni column;<br> 

6. PKA_alpah with pG-KJE8, from Ni column;<br> 

7. PKA_apha with pKJE7, from Ni column;<br> 

8. PKA-apha with pTf16, from Ni column; 

9. PKA-alpha with pG-Tf2, from Ni column; 

<br> 

<br> 

<br><br> --[[User:Ruili.su|Ruili.su]] 09:36, 13 April 2011 (EST) 

for PKA expression: pGro7 and pTf16 are good. 

<br> 

[[Image:MBP&NUSA 4-14.jpg]] 

<br> 

1. undigested MBP; 

2. digested MBP; 

3. undigested NUSA; 

4. digested NUSA<br> 

--[[User:Ruili.su|Ruili.su]] 09:38, 15 April 2011 (EST) 

.[[Image:VP16 purification 2 4 24 2011.jpg]] 

1. VP16, Morning elute<br> 

2. Vp16, Afternoon elute<br> 

3. Vp16, afternoon wash<br> 

4. pKA_alpha in pSpeedET with pTF16 chaperone<br> 

5. pKA_alpha in pSpeedET with pKJE7 chaperone<br> 

6. pKA_alpha in pSpeedET with pG-KJE8 chaperone<br> 

<br> 

[[Image:VP16 purification 4 24 2011.jpg]]<br> 

VP16 gel filtration HiLoad 16/60 column, TBS buffer, fration 30 to 35.<br> 

<br> 

[[Image:Vp16 western blot 4 24 11.jpg]] 

VP16 western blot, using anti-His tag mouse monoclonal antibody.<br> 

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<br> 

<br> 

<br> 

--[[User:Paul.xu|Paul.xu]] 12:38, 24 April 2011 (EST) <br> 

SDS-PAGE and western blot analysis of Ruili purified Vp16<br> 

[[Image:Vp16 purification 4 25 11.jpg]]<br> 

Western blot using mouse anti-MBP monoclonal antibody <br> 

[[Image:Vp16 westernblot-2 4 25 11.jpg]]<br> 

Sample #<br> 

&nbsp;MW ladder 10, 15, 25 (red), 35, 55, 70(red), 100, 130, 250. <br> 

2: vp16 -1 

3: vp16- OD280: 0.045 

4: VP16 1.29 

5: VP16 

6: empty 

7: MBP: OD280=0.23 

8: Morning elute 

9: Vp16 in centricon. 

So these VP16 proteins purified by Ruili are not VP16, they are MBP. 

--[[User:Paul.xu|Paul.xu]] 18:09, 25 April 2011 (EST) 

[[Category:Protein_Purification]]