Paul.xu at 04:39, 18 February 2019

2019-02-18T04:39:35Z

New page

== Protein Expression ==

2014/10/17 SHANYI

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

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

|
0.5 mM IPTG 

30oC 4.5h

| Ni beads 
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2g/500ml

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

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| 2 
| pET28b-HTNCre 
| 375 
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RossetaplySs

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0.5 mM IPTG

10ml 50x5020

RT O/N

| Ni beads 
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3.8g/50ml

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

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| 3 
| pT7A3-HTNCre 
| 1708 
|
Nico21(DE3)plysN

|
0.5 mM IPTG

30oC 4.5h

| Ni beads 
|

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

|-
| 4 
| pCold-HTNCre 
| 1823 
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Nico21(DE3)plysN

|
0.5 mM IPTG

RT 5.5h

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

|-
| 5 
| pBad24-HTNCre 
| 1010 
|
Nico21(DE3)plysN

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0.2%Arabinose

30oC 6h

| Ni beads 
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7.7g/1.25L

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

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| 6 
| pTriEx-HTNCre 
| 298 
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 BL21(DE3) pLysS

| 0.5 mM IPTG 30oC ?h 
| Ni beads 
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| 
| grow very slowly 
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| 7 
| pET28b-HTNCre 
| 375 
|   Nico21(DE3)plysN

| 0.5 mM IPTG 37oC 2h 
| Ni beads 
|

| 
| pellet from 8L purification 
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| 8 
| PageRuler Plus Prestained protein ladder 
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| 9 
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| rowspan="4" | HTNCre 
| 1708 
|   Nico21(DE3)plysN 
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| 10mM Imidazol wash flowthrough 
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| 10 
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| 1708 
|   Nico21(DE3)plysN 
| 0.5 mM IPTG 37oC 2h 
| Ni beads 
| rowspan="2" | 
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| 20mM Imidazol wash flowthrough 
|-
| 11 
| 
| 298 
|   BL21(DE3) pLysS 
| 0.5 mM IPTG 37oC 2h 
| Ni beads 
|  10mM Imidazol wash flowthrough 
|-
| 12 
| 
| 298 
|
BL21(DE3) pLysS

| 0.5 mM IPTG 37oC 2h 
| 
| 
| 
| 20mM Imidazol wash flowthrough 
|}

10/1/2018

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

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

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

2/17/19

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

Cre expression screening - Revision history

Paul.xu at 04:39, 18 February 2019