1625 pT7A3-MBP-(3c)-KEAP1

Cloning of 1625 pT7A3-MBP-(3c)-KEAP1

Purpose: Cloning of 1625 pT7A3-MBP-(3c)-KEAP1

Date: 2013-1-24---2013-1- 28

Notebook:0

Page:

Clone ID:1625

Experimenters: yingying

Results

PCR

1/27/2013

Colony Screening

Date: 2013-1-

Make miniprep and digest with

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Cloning of 1625 pT7A3-MBP-(3c)-KEAP1

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

PCR

digest pcr product with Nde1 + Xho1, clone into Nde1 + Xho1 digested 1565,pT7A3-MBP-(3C)-sfGFP2

Colony Screening

Date: 2013-1-31

Make miniprep and digest with Nde1 + Xho1

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Protein Expression

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

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