1613-pET28-cold-GST-3C
Cloning of 1613-pET28-cold-GST-3C
[edit]Purpose: Cloning of 1613-pET28-cold-GST-3C
Date: 2013-1-10---2013-1-14
Notebook:0
Page:
Clone ID:1613
Experimenters: yingying
Results
[edit]cloning
[edit]
digest PCR product with Pst1 + Xho1, clone into V20 pET28-cold-GST
Colony Screening
[edit]Date: 2013-1-14
Make miniprep and digest with Pst1 + Xho1
Result intepretation/Summary
[edit]Conclusion
[edit]Protein Expression
[edit]2013/2/12
Load GST-3C protein (clone: 1613, pET28-cold-GST-3C) purified from Ni column to 1 ml HiTrap GST column, wash with 15 ml of 25 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA. Elute with 10 mM Glutathione in 20 mMTris-HCl, pH7.5, 5 CV, 2 ml per fraction. Use 10 ul for SDS-PAGE
| Lane |
Purification |
| M |
Fermentas |
| 1 |
Eluate from Glutathione beads Fraction 1, 1st column |
| 2 |
Eluate from Glutathione beads Fraction 2, 1st column |
| 3 |
Eluate from Glutathione beads Fraction 3, 1st column |
| 4 |
Eluate from Glutathione beads Fraction 1, 2nd column |
| 5 |
Eluate from Glutathione beads Fraction 2, 2nd column |
| 6 |
Eluate from Glutathione beads Fraction 3, 2nd column |
| 7 |
FT, 1st column |
| 8 |
FT, 2nd column |
| 9 |
wash, 1st column |
| 10 |
load |
| 11 |
load |
2013/2/13
Load GST-3C protein (clone: 1613, pET28-cold-GST-3C) purified from Ni column to 1 ml HiTrap GST column, wash with 15 ml of 25 mM Tris-HCl, pH6.9, 600 mM NaCl, 1 mM b-ME, Elute with 10 mM Glutathione in 20 mMTris-HCl, pH7.5, 5 CV, 1 ml per fraction, discard the first fraction (1 ml, no protein). Use 10 ul for SDS-PAGE
| Lane |
Purification |
| M |
Fermentas |
| 1 |
Eluate from Glutathione beads Fraction 1, 1st column |
| 2 |
Eluate from Glutathione beads Fraction 2, 1st column |
| 3 |
Eluate from Glutathione beads Fraction 3, 1st column |
| 4 |
Eluate from Glutathione beads Fraction 1, 2nd column |
| 5 |
Eluate from Glutathione beads Fraction 2, 2nd column |
| 6 |
Eluate from Glutathione beads Fraction 3, 2nd column |
| 7 |
FT, 1st column |
| 8 |
Wash (combined, 30 ul) |
| 9 |
pT7A3-MBP-i3C, 1554, 4 ml ,NiCO21(DE3)pLysN, 0.2 mM IPTG 25 oC o/n, from Ni beads.(only MBP is expressed) |
| 10 |
pT7A3-6His-GST-i3C, 1601, 4 ml, NiCO21(DE3)pLysN, 0.2 mM IPTG 25 oC o/n, from Ni beads.(only GST is expressed) |
2013/2/21
| Lane |
Name |
Clone ID |
Host |
Induction |
Purification |
| M |
Invitrogen SeeBlue plus2 |
||||
| 1~7 |
pET28-cold-GST-3C |
1613 |
BL21(DE3)Star, pRare2-pLys, 8L |
0.2 mM IPTG 25 oC o/n |
SEC on TSK-Gel |
| 8 |
pT7A3-MBP-(3c)-NRF2 |
1661 |
4 ml, BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
| 9&11 |
pCold-GST-(3c)-KEAP1 |
1625 |
8L BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |