Editing 1613-pET28-cold-GST-3C (section)

== Protein Expression<br>  ==

2013/2/12<br> 

Load GST-3C protein (clone: 1613, pET28-cold-GST-3C) purified from Ni column to 1 ml HiTrap GST column, wash with 15 ml of 25 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA. Elute with 10 mM Glutathione in 20 mMTris-HCl, pH7.5, 5 CV, 2 ml per fraction. Use 10 ul for SDS-PAGE<br> 

[[Image:GST-3C purification 2013 2 12.jpg]]<br> 

{| width="381" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br> 
| Purification<br>
|-
| M<br> 
| Fermentas <br>
|-
| 1<br> 
| Eluate from Glutathione beads Fraction 1, 1st column<br>
|-
| 2<br> 
| Eluate&nbsp; from Glutathione beads Fraction 2, 1st column<br>
|-
| 3<br> 
| Eluate from Glutathione beads Fraction 3, 1st column<br>
|-
| 4<br> 
| Eluate from Glutathione beads Fraction 1, 2nd column<br>
|-
| 5<br> 
| Eluate&nbsp; from Glutathione beads&nbsp; Fraction 2, 2nd column<br>
|-
| 6<br> 
| Eluate from Glutathione beads Fraction 3, 2nd column<br>
|-
| 7<br> 
| FT, 1st column<br>
|-
| 8<br> 
| FT, 2nd column<br>
|-
| 9<br> 
| wash, 1st column<br>
|-
| 10<br> 
| load<br>
|-
| 11<br> 
| 
load<br> 

|}

2013/2/13<br> 

Load GST-3C protein (clone: 1613, pET28-cold-GST-3C) purified from Ni column to 1 ml HiTrap GST column, wash with 15 ml of 25 mM Tris-HCl, pH6.9, 600 mM NaCl, 1 mM b-ME, Elute with 10 mM Glutathione in 20 mMTris-HCl, pH7.5, 5 CV, 1 ml per fraction, discard the first fraction (1 ml, no protein). Use 10 ul for SDS-PAGE<br> 

[[Image:GST-3C purification 2013 2 13.jpg]]<br> 

{| width="381" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br> 
| Purification<br>
|-
| M<br> 
| Fermentas <br>
|-
| 1<br> 
| Eluate from Glutathione beads Fraction 1, 1st column<br>
|-
| 2<br> 
| Eluate&nbsp; from Glutathione beads Fraction 2, 1st column<br>
|-
| 3<br> 
| Eluate from Glutathione beads Fraction 3, 1st column<br>
|-
| 4<br> 
| Eluate from Glutathione beads Fraction 1, 2nd column<br>
|-
| 5<br> 
| Eluate&nbsp; from Glutathione beads&nbsp; Fraction 2, 2nd column<br>
|-
| 6<br> 
| Eluate from Glutathione beads Fraction 3, 2nd column<br>
|-
| 7<br> 
| FT, 1st column<br>
|-
| 8<br> 
| Wash (combined, 30 ul)<br>
|-
| 9<br> 
| pT7A3-MBP-i3C, 1554, 4 ml ,NiCO21(DE3)pLysN, 0.2 mM IPTG 25 oC o/n, from Ni beads.(only MBP is expressed)<br>
|-
| 10<br> 
| pT7A3-6His-GST-i3C, 1601, 4 ml, NiCO21(DE3)pLysN, 0.2 mM IPTG 25 oC o/n, from Ni beads.(only GST is expressed)
|}

<br> 

2013/2/21<br> 

[[Image:2_21_2013_protein_purification.jpg]]<br> 

<br> 

{| width="684" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br> 
| Name<br> 
| Clone ID<br> 
| Host<br> 
| Induction<br> 
| Purification<br>
|-
| M<br> 
| Invitrogen SeeBlue plus2<br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 1~7<br> 
| pET28-cold-GST-3C<br> 
| 1613<br> 
| 
BL21(DE3)Star, pRare2-pLys, 8L<br>

| 0.2 mM IPTG 25 oC o/n<br> 
| SEC on TSK-Gel<br>
|-
| 8<br> 
| pT7A3-MBP-(3c)-NRF2 <br> 
| 1661<br> 
| 
4 ml, 

BL21(DE3)Star, pRare2-pLysS <br> 

| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|-
| 9&amp;11<br> 
| pCold-GST-(3c)-KEAP1<br> 
| 1625<br> 
| 
8L 

BL21(DE3)Star, pRare2-pLysS <br> 

| 0.2 mM IPTG 25 oC o/n<br> 
| Ni beads<br>
|}

[[Category:Cloning]]