Vp16-purification

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

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

1. NUSA, undigested, from Ni column;

2. MBP, undigested, from Ni column;

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;

5. PKA_alpha with pGro7, from Ni column;

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

7. PKA_apha with pKJE7, from Ni column;

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

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

--Ruili.su 09:36, 13 April 2011 (EST)

for PKA expression: pGro7 and pTf16 are good.

1. undigested MBP;

2. digested MBP;

3. undigested NUSA;

4. digested NUSA

--Ruili.su 09:38, 15 April 2011 (EST)

.

1. VP16, Morning elute

2. Vp16, Afternoon elute

3. Vp16, afternoon wash

4. pKA_alpha in pSpeedET with pTF16 chaperone

5. pKA_alpha in pSpeedET with pKJE7 chaperone

6. pKA_alpha in pSpeedET with pG-KJE8 chaperone

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

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

--Paul.xu 12:38, 24 April 2011 (EST)

SDS-PAGE and western blot analysis of Ruili purified Vp16

Western blot using mouse anti-MBP monoclonal antibody

Sample #

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

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.

--Paul.xu 18:09, 25 April 2011 (EST)