Editing 1185-pDB.His.MBP-Int (section)

=== == ===

10/21/19

Purification from 2 L culture. Induce at 18 oC o/n, 0.5 mM IPTG

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| Sample, fraction #
| Note
| Note
|-
| 1
| 1185, #37
| looks like MBP, truncated, maybe int is not expressed
| Cell lysed and regrow,&nbsp;
|-
| 2
| 1185, #38
| looks like MBP, truncated, maybe int is not expressed<br/>
| truncated, only MBP
|-
| 3
| 1078
| Ni-->Q, elute using 0.05-->1.5 M NaCl gradient<br/>
| <br/>
|-
| 4
| 1078
| Ni-->Q, elute using 0.05-->1.5 M NaCl gradient<br/>
| <br/>
|-
| 5
| 1186 , #5
| Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
| has expression
|-
| 6
| 1186, #8
| Ni-->Q, elute using 0.05-->1.5 M NaCl gradient<br/>
| has expression<br/>
|-
| 7
| 1186, #6
| washed from Ni using 30 mM Imidazole
| has expression<br/>
|-
| 8
| 1186, #7
| washed from Ni using 30 mM Imidazole<br/>
| has expression<br/>
|-
| 9
| 1185, #12
| Ni-->Q, elute using 0.05-->1.5 M NaCl gradient<br/>
| <br/>
|-
| 10
| 1185, #16<br/>
| Ni-->Q, elute using 0.05-->1.5 M NaCl gradient<br/>
| <br/>
|-
| 11
| 1185, #17<br/>
| Ni-->Q, elute using 0.05-->1.5 M NaCl gradient<br/>
| <br/>
|-
| M
| PageRuler Plus<br/>
| 250, 130, 00,&nbsp;<span style="color: rgb(255, 0, 0);">70</span>, 55, 35,&nbsp;<span style="color: rgb(255, 0, 0);">25</span>, 15, 10<br/>
| <br/>
|}





[[File:1185 1078 1186 purification.jpg|RTENOTITLE]]

Purification of 1185

expression host: Nico21(DE3)plysN, LB with 1X 5052, 0.5 mM IPTG, 17 oC o/n

[[File:1185 expression truncated.jpg|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| ID
| Name
| Fraction
|-
| 1
| 1186&nbsp;
| MBP-Xis
| Lot 173
|-
| 2
| 1186
| MBP-Xis<br/>
| Lot 172
|-
| 3
| 1185
| MBP (truncated from Int exp)
| Lot 171
|-
| 4
| 1185
| MBP (truncated from Int exp)
| Lot 174&nbsp; elute from Q-SP, fraction 18
|-
| 5
| <br/>
| <br/>
| Lot 174&nbsp; elute from Q-SP,&nbsp;F 19
|-
| 6
| <br/>
| <br/>
| Lot 174&nbsp; elute from Q-SP,&nbsp;F 20
|-
| 7
| <br/>
| <br/>
| Lot 174&nbsp; elute from Q-SP,&nbsp;F 21
|-
| 8
| <br/>
| junk, discard
| Lot 174&nbsp; elute from Q-SP,&nbsp;F25
|-
| 9
| <br/>
| junk, discard
| Lot 174&nbsp; elute from Q-SP,&nbsp;F26
|-
| 10
| <br/>
| <br/>
| Lot 174&nbsp;FT on&nbsp;Q-SP F7
|-
| 11
| <br/>
| <br/>
| Lot 174&nbsp;FT on&nbsp;Q-SP F27<br/>
|-
| M
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|}

11/1/2019

Use Nico21(DE3)pRare2pLysS, 15 ml, induced with 1 mM IPTG, 37 oC 5 hours

cre in Nico21(DE3)pLysN, (from freezer, not freshly transformed) 8L, induce 37 oC 6 hours, 0.15% arabinose., use 50 ml Ni column

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| 1
| <br/>
| MW
| Expression
|-
| 1
| 1078
| pET28b-scIHF2, soluble<br/>
| 22.8
| N
|-
| 2
| 406
| <span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pET28b-Int</span>, soluble<br/>
| 42.5
| N
|-
| 3
| 1185
| <span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pDB.His.MBP-Int</span>, soluble<br/>
| 83.7
| Y
|-
| 4
| 2640
| pCold-MBP-(Ent-TEV)-scIHF, soluble<br/>
| 66.9
| N
|-
| 5
| 2640
| pCold-MBP-(Ent-TEV)-scIHF, inclusion body<br/>
| 66.9<br/>
| Y
|-
| 6~12
| 1010&nbsp;
| Cre,&nbsp;<br/>
| <br/>
| could be low
|-
| 13
| 1078
| pET28b-scIHF2<br/>
| 22.8<br/>
| N
|-
| 14
| 406
| <span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pET28b-Int</span><br/>
| 42.5<br/>
| N
|-
| M
| <br/>
| PageRuler Plus
| <br/>
| <br/>
|}

[[File:1185 2640 expression.jpg|RTENOTITLE]]
[[Category:Cloning]]