2115 1996 1592 1315

2014/3/

lane

1

2

3

4

5

6

7

8

clone ID

2115, MW is 52.8 kDa pT7A3-iFLP

PageRuler Plus Prestained protein ladder

pL6H-LambdaExo

pT7A3-Ecoli-Topo_I

pT7A3-MthRnl

host

Nico21DE3plysN

Nico21DE3plysN

induction

0.1 mM IPTG 25 oC o/n

0.2 mM IPTG 25 oC o/n

purification

Ni beads

Ni beads

lysis buffer

buffer A ---- 99oC 10min

buffer A

buffer A (containing 1M Nacl)

purification lysis buffer(containing NP40+1M Nacl)

expression screening lysis buffer(containing 4M Urea+1% SDS)

MW

pI/Mw: 9.76 / 52.8

6.06 / 27.1

8.68/97.3

5.81 / 45.3

2115 pT7A3-iFLP----8L, Nico(DE3)plysN, 0.1mM IPTG RT induce overnight, 34.1g
use 5ml induced culture to chose the best lysis buffer
5. expression screening lysis buffer(containing 4M Urea+1% SDS) -------clear----have expression
4. purification lysis buffer(containing NP40+1M Nacl)-------------white pellet-------no expression
3. buffer A (containing 1M Nacl)-------------white pellet-------no expression
2. buffer A -------------white pellet-------no expression
1. buffer A ---- 99oC 10min---------white pellet-------no expression

then add buffer A containing 1M Nacl to pellets
1.add Urea to final concentration of 4M------no change
2.add SDS to final concentration of 1 % -------half of the pellet dissolved
3.add Urea to final concentration of 4M and add SDS to final concentration of 1 % ------ all of the pellet dissolved
4.add Arginine to final concentration of 0.1M, add glycerol to to final concentration of 10 %, add triton-x100 to final concentration of 0.5%-----no change
add lysis buffer (containing DnaseI+ lysozyme) to pellet-----37oC 2hours----no change