1707-pT7A3-MBP-(E3C)-p53wt

Cloning of 1707-pT7A3-MBP-(E3C)-p53wt

Purpose: Cloning of 1707-pT7A3-MBP-(E3C)-p53wt

Date: 2013-3-20---2013-3-22

Notebook:0

Page:

Clone ID:1707

Experimenters: yingying

Results

cloning

Colony Screening

Date: 2013-3-22

Make miniprep and digest with Nhe1 + EcoR1

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Result intepretation/Summary

• &amp;amp;lt;br&amp;amp;gt;

Protein screening

2013/3/30

lane	name	clone ID	host	induction	purification		MW
1	pT7A3-T5-Exo	1567	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads		21KD/5.95
2	pCold-TF-(3c)	1614	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads	55.9KD/5.19
3	pET28b-Nde1	1673	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads		44KD/8.41
4	pTCold-MBP-(3C)	1677	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads		44KD
5	pCold-C(3C)-MBP-6H	1692	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads		46KD/5.77
6	pT7A3-wtSM2	1694	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads, should use CBD beads		33KD/6.30
7	pCold-MBP-(3C)-TEV	1696	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads		72KD/6.04
8	pCold-GST-(3c)-wt3C	1697	4 ml,NiCO21(DE3)pLysN	0.2 mM IPTG 25 oC o/n	Ni beads		21KD/8.47
9	pT7A3-MBP-(E3C)-p53wt	1707	4 ml, BL21(DE3)Star, pRare2-pLysS	0.2 mM IPTG 25 oC o/n	Ni beads		88KD/5.76
10	pET28b-hINFA2	1711	4 ml, BL21(DE3)Star, pRare2-pLysS	0.2 mM IPTG 25 oC o/n	Ni beads		21KD/5.95

Protein Purification

1. Step gradient, linear gradient on 5 ml HisTrap column

SDS-PAGE: 2013/4/5

lane	1	2	3	4	5	6	7	8	9	10
clone ID	1696-pCold-MBP-(3C)-TEV					1707-pCold-MBP-(3C)-TEV		1567pT7A3-T5-Exo	1673-pET28b-Nde1	1697-pCold-GST-(3c)-wt3C
host	Nico21DE3plysN					BL21DE3pRare2pLysS		Nico21DE3plysN	Nico21DE3plysN	Nico21DE3plysN
induction	0.2 mM IPTG 25 oC o/n					0.2 mM IPTG 25 oC o/n		0.2 mM IPTG 25 oC o/n	0.2 mM IPTG 25 oC o/n	0.2 mM IPTG 25 oC o/n
purification	5ml HisTrap HP					5ml HisTrap HP		Ni beads	Ni beads	Ni beads
MW	72KD/6.04					88KD/5.76		33KD/5.22	44KD/8.41	21KD/8.47

2. Gel filtration on TSK 250 gel filtration.

Buffer:

SDS-PAGE: 2013/4/9

Peak 1 and 2: p53-MBP fusion and MBP (fraction 17 to 21)

Peak 3: MBP.

concentrate peak 1 (fraction 17 ~21) using spin concentator

Combine MBP fractions stored in cold room.

3. 3C digestion

4. Load on amylose column. Collect flow through. Concentrate protein, store in a buffer (xxx, 10% glycerol) at -80oC. Lot #: