Editing Cre expression screening (section)

== Protein Expression ==

2014/10/17<br/>SHANYI

[[File:375 375 1708 1823 1010 298 375p.jpg|RTENOTITLE]]



{| width="993" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br/>
| Name<br/>
| Protein Name<br/>
| Clone ID<br/>
| Host<br/>
| Induction<br/>
| Purification<br/>
| pI/Mw<br/>
| express<br/>
| note<br/>
|-
| 1<br/>
| pET28b-HTNCre<br/>
| rowspan="7" | <br/><br/><br/><br/><br/><br/><br/>cre
| 375<br/>
| 
Nico21(DE3)plysN

| 
0.5 mM IPTG&nbsp;

30oC 4.5h

| Ni beads<br/>
| 


| <br/>
| 
2g/500ml

suspend in 40ml BufferA,take 1ml to do protein expression screening

|-
| 2<br/>
| pET28b-HTNCre<br/>
| 375<br/>
| 
RossetaplySs

| 
0.5 mM IPTG

10ml 50x5020

RT O/N

| Ni beads<br/>
| 


| <br/>
| 
3.8g/50ml

suspend in 45ml BufferA,take 1ml to do protein expression screening

|-
| 3<br/>
| pT7A3-HTNCre<br/>
| 1708<br/>
| 
Nico21(DE3)plysN

| 
0.5 mM IPTG

30oC 4.5h

| Ni beads<br/>
| 


| <br/>
| 3g/500ml
suspend in 45ml BufferA,take 1ml to do protein expression screening

|-
| 4<br/>
| pCold-HTNCre<br/>
| 1823<br/>
| 
Nico21(DE3)plysN

| 
0.5 mM IPTG

RT 5.5h

| Ni beads<br/>
| <br/>
| <br/>
| 3g/500ml
suspend in 45ml BufferA,take 1ml to do protein expression screening

|-
| 5<br/>
| pBad24-HTNCre<br/>
| 1010<br/>
| 
Nico21(DE3)plysN

| 
0.2%Arabinose

30oC 6h

| Ni beads<br/>
| 


| <br/>
| 
7.7g/1.25L

suspend in 110ml BufferA,take 1ml to do protein expression screening

|-
| 6<br/>
| pTriEx-HTNCre<br/>
| 298<br/>
| 
&nbsp;BL21(DE3) pLysS

| 0.5 mM IPTG 30oC&nbsp;?h<br/>
| Ni beads<br/>
| 


| <br/>
| grow very slowly<br/>
|-
| 7<br/>
| pET28b-HTNCre<br/>
| 375<br/>
| &nbsp; Nico21(DE3)plysN


| 0.5 mM IPTG 37oC 2h<br/>
| Ni beads<br/>
| 


| <br/>
| pellet from 8L purification<br/>
|-
| 8<br/>
| PageRuler Plus Prestained protein ladder<br/><br/>
| <br/>
| <br/>
| &nbsp;<br/>
| <br/>
| <br/>
| 


| <br/>
| <br/>
|-
| 9<br/>
| <br/>
| rowspan="4" | <br/><br/>HTNCre<br/>
| 1708<br/>
| &nbsp; Nico21(DE3)plysN<br/>
| <br/>
| <br/>
| 


| <br/>
| 10mM Imidazol wash flowthrough<br/>
|-
| 10<br/>
| <br/><br/>
| 1708<br/>
| &nbsp; Nico21(DE3)plysN<br/>
| 0.5 mM IPTG 37oC 2h<br/>
| Ni beads<br/>
| rowspan="2" | <br/>
| <br/>
| 20mM Imidazol wash flowthrough<br/>
|-
| 11<br/>
| <br/>
| 298<br/>
| &nbsp; BL21(DE3) pLysS<br/>
| 0.5 mM IPTG 37oC 2h<br/>
| Ni beads<br/>
| &nbsp;10mM Imidazol wash flowthrough<br/>
|-
| 12<br/>
| <br/>
| 298<br/>
| 
BL21(DE3) pLysS

| 0.5 mM IPTG 37oC 2h<br/>
| <br/>
| <br/>
| <br/>
| 20mM Imidazol wash flowthrough<br/>
|}



10/1/2018

[[File:Cre 1 10 1 18.jpg|RTENOTITLE]]



{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane<br/>
| &nbsp;Name<br/>
| Protein Name&nbsp;<br/>
| Clone ID<br/>
| Host<br/>
| Induction<br/>
| Purification<br/>
| &nbsp;pI/Mw<br/>
| &nbsp;express<br/>
| &nbsp; note<br/>
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[[File:Cre 2 10 1 18..jpg|RTENOTITLE]]



{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane<br/>
| &nbsp;Name<br/>
| Protein Name&nbsp;<br/>
| Clone ID<br/>
| Host<br/>
| Induction<br/>
| Purification<br/>
| &nbsp;pI/Mw<br/>
| &nbsp;express<br/>
| &nbsp; note<br/>
|-
| 1
| M
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 2
| Original lysis cell fluid
| Cre
| 1708
| NIco21
| 1mM IPTG,o/n,
| Ni beads
| <br/>
| <br/>
| <br/>
|-
| 3
| Ni flow through
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 4
| From Buffer A Wash
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 5
| From immedizole buffer A wash
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 6
| Ni elution
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 7
| Q flowthrough
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 8
| SP Flow through
| <br/>
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| <br/>
| <br/>
|-
| 9
| From SP washing buffer wash
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|-
| 10
| SP Elution
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 11
| From Nico 21
| cre
| 1010
| Nico21
| <br/>
| Ni Beads
| <br/>
| <br/>
| <br/>
|-
| 12
| From BL 21
| cre
| 1010
| BL21 star
| <br/>
| Ni Beads
| <br/>
| <br/>
| <br/>
|}

2/17/19

[[File:2115 2108 1708 expression.jpg]][[Category:Protein Purification]]