Excellgen at 21:16, 6 November 2014

2014-11-06T21:16:55Z

New page

== Protein Expression ==

2014/10/29 SHANYI

[[Image:Cre1708 2367 2368.jpg]] 

{| width="832" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| Name 
| Protein Name 
| Clone ID 
| Host 
| Induction 
| Purification 
| pI/Mw 
| express 
|-
| 1 
| rowspan="6" | pT7A3-HTNCre 
| 
| rowspan="6" | 1708 
| rowspan="2" | 1L Nico21(DE3)plysN 
|
0.5 mM IPTG  

37oC 4.75h 

| Ni beads 
|
 

| 
|-
| 2 
| 
| 0.5 mM IPTG
37oC 4.75h

 

| Ni beads 
|
 

| wash with 35mM Imidazole 
|-
| 3 
| 
| rowspan="2" | 1L Nico21(DE3)plysN
| 0.5 mM IPTG
30oC 4.75h

 

| Ni beads 
|
 

| 
|-
| 4 
| 
| 0.5 mM IPTG
30oC 4.75h

 

| Ni beads 
| 
| wash with 35mM Imidazole 
|-
| 5 
| 
| 1L Nico21(DE3)plysN 
| 0.5 mM IPTG(ours)
RT O/N 

| Ni beads 
|
 

| 
|-
| 6 
| 
|
1L Nico21(DE3)plysN 

| 0.5 mM IPTG
RT O/N 

| Ni beads 
|
 

| 
|-
| 7 
| PageRuler Plus
protein ladder 

| 
| 
|   
| 
| 
|
 

| 
|-
| 8 
| pCold-MBP-(Ent-TEV)-HCMVUL98 
| 
| 2367 
|   15 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG
RT O/N 

| Ni beads 
|
 

| 
|-
| 9 
| pCold-MBP-(Ent-TEV)-HCMVUL98 
| 
| 2367 
|   15 ml,
Tuner(DE3)plysN-tev 

| 0.2 mM IPTG
RT O/N

 

| Ni beads 
|
 

| 
|-
| 10 
| pT7A3-MBP-(TEV)-HCMVUL98 
| rowspan="2" | 
| 2368 
|
15 ml,

Nico21(DE3)plysN 

| 0.2 mM IPTG
RT O/N 

| Ni beads 
| 110 
| 
|-
| 11 
| pT7A3-MBP-(TEV)-HCMVUL98 
| 2368 
|   15 ml,
Tuner(DE3)plysN-tev 

| 0.2 mM IPTG
RT O/N 

| Ni beads 
| 67+42 
| 
|-
| 12 
| pCold-MBP-(Ent-TEV)-HCMVUL98 
| 
| 2367 
| 5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG
RT O/N 

| Ni beads 
| 
| pellet from lane8 
|}

2014/10/30 

combine cre lysate from 4L 1708 Nico21(DE3)plysN 

purifying using 20ml Ni column (new Ni beads packed in XK 16/20 Column),lysisbuffer and buffer A are NaPO4 buffer PH7.4 

after loading sample. the column turns white

wash with 5cv 5mM Imidazole , then elute, when the peak comes out, stop FPLC at Imidazole concentration is 150mM

FPLC result: method run10/31/2014,5:08:20AM

[[Image:Cre1708purification 20141031.jpg]] 

lane1:600ul lysate (purified with 75ul screening Ni beads ) 

lane2:Ni column flowthrough(purified with 75ul screening Ni beads ) 

lane3-7:protein eluted from Ni column 

lane8:PageRuler Plus protein ladder 

lane9: protein eluted from Ni column combined together 30ul 

lane10: sp ff flowthrough 30ul 

lane11: protein eluted from sp ff(250-1M NaCl) 

lane12: protein eluted from sp ff(2M NaCl 100mM Arginine 20mM Tris-Hcl) 

[[Category:Protein_Purification]]

Cre1708 expression screening 2367 2368 2014 10 29 - Revision history

Excellgen at 21:16, 6 November 2014