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