Editing Cre1708 expression screening 2367 2368 2014 10 29

== Protein Expression  ==

2014/10/29<br> SHANYI 

[[Image:Cre1708 2367 2368.jpg]]<br> 

{| width="832" 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>
|-
| 1<br> 
| rowspan="6" | <br> pT7A3-HTNCre <br> <br> 
| <br> 
| rowspan="6" | <br> 1708 <br> <br> 
| rowspan="2" | 1L<br>Nico21(DE3)plysN<br> <br> 
| 
0.5 mM IPTG&nbsp;<br> 

37oC 4.75h<br> 

| Ni beads<br> 
| 
<br> 

| <br>
|-
| 2<br> 
| <br> 
| 0.5 mM IPTG 
37oC 4.75h 

<br> 

| Ni beads<br> 
| 
<br> 

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

<br> 

| Ni beads <br> 
| 
<br> 

| <br>
|-
| 4<br> 
| <br> 
| 0.5 mM IPTG 
30oC 4.75h 

<br> 

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

| Ni beads <br> 
| 
<br><br> 

| <br>
|-
| 6<br> 
| <br> 
| 
1L<br>Nico21(DE3)plysN<br> 

| 0.5 mM IPTG 
RT O/N<br> 

| Ni beads<br> 
| 
<br> 

| <br>
|-
| 7<br> 
| PageRuler Plus 
protein ladder <br> 

| <br> 
| <br> 
| &nbsp; <br> 
| <br> 
| <br> 
| 
<br> 

| <br>
|-
| 8<br> 
| pCold-MBP-(Ent-TEV)-HCMVUL98<br><br> 
| <br> 
| 2367<br> 
| &nbsp; 15 ml, 
Nico21(DE3)plysN<br> 

| 0.2 mM IPTG 
RT O/N<br> 

| Ni beads<br> 
| 
<br> 

| <br>
|-
| 9<br> 
| pCold-MBP-(Ent-TEV)-HCMVUL98<br> 
| <br> 
| 2367<br> 
| &nbsp; 15 ml, 
Tuner(DE3)plysN-tev<br> 

| 0.2 mM IPTG 
RT O/N 

<br> 

| Ni beads<br> 
| 
<br> 

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

Nico21(DE3)plysN<br> 

| 0.2 mM IPTG 
RT O/N<br> 

| Ni beads<br> 
| 110<br> 
| <br>
|-
| 11<br> 
| pT7A3-MBP-(TEV)-HCMVUL98<br> 
| 2368<br> 
| &nbsp; 15 ml, 
Tuner(DE3)plysN-tev<br> 

| 0.2 mM IPTG 
RT O/N<br> 

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

| 0.2 mM IPTG 
RT O/N<br> 

| Ni beads<br> 
| <br> 
| pellet from lane8<br>
|}

2014/10/30<br> 

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

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

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]]<br> 

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

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

lane3-7:protein eluted from Ni column<br> 

lane8:PageRuler Plus protein ladder <br> 

lane9: protein eluted from Ni column combined together 30ul<br> 

lane10: sp ff flowthrough 30ul<br> 

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

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

[[Category:Protein_Purification]]