1676 pCold-MBP-(3C)-KEAP1
Subcloning of KEAP1 into pCold-MBP-(3C)
[edit]Purpose: Subcloning of KEAP1 from 1625 pT7A3-MBP-(3c)-KEAP1 to 1611 pCold-MBP-(3C)
Date: 2013-2-26---2013-2-28
Notebook:
Page:
Clone ID: 1676
Experimenters: Yingying
Results
[edit]Subcloning
[edit]Date:
Vector: 1611 pCold-MBP-(3C)
Parent: 1625 pT7A3-MBP-(3c)-KEAP1
Enzymes: Nde1 + Xho1
Colony Screening
[edit]Date: 2013-2-28
Nde1 + Xho1
Protein Expression
[edit]2013/3/5
| Lane |
Name |
Clone ID |
Host |
Induction |
Purification |
| M |
Fermentas PageRuler Plus |
||||
| 1 |
pT7A3-MBP-(3c)-NRF2 |
1661 clone 1 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 2 |
pT7A3-MBP-(3c)-KEAP1 |
1661 Clone 2 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 3 |
pCold-GST-(3c)-NRF2 |
1674 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads (wrong, should use Glutathione agarose beads, no his tag) |
| 4 |
pCold-GST-(3c)-NRF2 |
1674 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads (wrong, should use Glutathione agarose beads, no his tag) |
| 5 |
pCold-MBP-(3C)-NRF2 |
1683 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 6 |
pCold-MBP-(3C)-NRF2 |
1683 |
4 ml,BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 7 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
4 ml,BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 8 |
pCold-MBP-(3C)-KEAP1 |
1676 |
4 ml,BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 9 | |||||
| 10 | Purified MBP |
Result intepretation/Summary
[edit]
Conclusion
[edit]Protein expression of KEAP1 clones
[edit]| clone ID |
expression |
ge image |
|
| 1625,pT7A3-MBP-(3c)-KEAP1 |
yes |
1625 1626 1656 |
|
| 1656,pCold-GST-(3c)-KEAP1 |
need to use GST beads |
1625 1626 1656 |
|
| 1676,pCold-MBP-(3C)-KEAP1 |
yes |
1661 1674 1683 |
|
| 1701,pMAL-c5E-KEAP1 |
yes |
||
| 1720,pT7A3-MBP-(E3C)-Keap1 |
no |
626 1717 1719 1720 |
|