1625 pT7A3-MBP-(3c)-KEAP1
Cloning of 1625 pT7A3-MBP-(3c)-KEAP1
[edit]Purpose: Cloning of 1625 pT7A3-MBP-(3c)-KEAP1
Date: 2013-1-24---2013-1- 28
Notebook:0
Page:
Clone ID:1625
Experimenters: yingying
Results
[edit]PCR
[edit]
1/27/2013
Colony Screening
[edit]Date: 2013-1-
Make miniprep and digest with
Result intepretation/Summary
[edit]Conclusion
[edit]
Cloning of 1625 pT7A3-MBP-(3c)-KEAP1
[edit]Purpose: Cloning of 1625 pT7A3-MBP-(3c)-KEAP1
Date: 2013-1-29---2-13-1-31
Notebook:0
Page:
Clone ID:1625
Experimenters: yingying
Results
[edit]PCR
[edit]
digest pcr product with Nde1 + Xho1, clone into Nde1 + Xho1 digested 1565,pT7A3-MBP-(3C)-sfGFP2
Colony Screening
[edit]Date: 2013-1-31
Make miniprep and digest with Nde1 + Xho1
Result intepretation/Summary
[edit]Conclusion
[edit]
Protein Expression
[edit]2013/2/11
| Lane |
Name |
Clone ID |
Host |
Induction |
Purification |
| M |
Invitrogen SeeBlue plus2 |
||||
| 1 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 2 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 3 |
pCold-GST-(3c)-KEAP1 |
1656 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads (wrong, should use Glutathione agarose beads) |
| 4 |
pCold-GST-(3c)-KEAP1 |
1656 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads (wrong, should use Glutathione agarose beads) |
| 5 |
pET28-cold-NRF2 |
1626 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 6 |
pT7A3-MBP-i3C |
1554 |
4 ml,NiCO21(DE3), pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 7 |
pT7A3-6His-GST-i3C |
1601 |
4 ml,NiCO21(DE3), pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 8 |
2/24/2013
| Lane |
Name |
Clone ID |
Host |
Induction |
Purification |
| M |
Invitrogen SeeBlue plus2 |
||||
| 1 |
pCold-GST-(3c)-NRF2 |
1674 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Glutathione beads |
| 2 |
pCold-GST-(3c)-NRF2 |
1674 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Glutathione beads |
| 3 |
pCold-GST-(3c)-NRF2 |
1674 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Glutathione beads |
| 4 |
pCold-GST-(3c)-NRF2 |
1674 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
uninduced |
Glutathione beads |
| 5 |
pET28-cold-GST-3C |
1613 |
8L, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, then gel filtration, Lot # 20130223 |
| 6 |
pET28-cold-GST-3C |
1613 |
8L,BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, then gel filtration, Lot # 20130224 |
| 7~9 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
8L BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, then amylose beads |
| 10~11 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
8L BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, then amylose beads |
2/25/2013
| Lane |
Name |
Clone ID |
Host |
Induction |
Purification |
| 1~5 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
8L, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, amylose beads, gel filtration, fraction # 39~43, this peak has 6His-MBP-KEAP1 fusion. |
| 6 |
Fermentas protein marker |
250, 130, 100, 70(red), 55, 35, 25(red), 15, 10 (bottom) | |||
| 7~12 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
8L, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, amylose beads, gel filtration, fraction # 65-70 (peak #2), this is MBP |
| 13 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
8L, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, amylose beads |
| 14 |
pT7A3-MBP-(3c)-KEAP1 |
1625 |
8L, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads, amylose beads, digest with GST-3C protease, Kaep1 without 6His-MBP fusion should be 70KDa (top Red band, fermentas protein marker) |
| 15 |
Invitrogen protein marker |
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 |
|