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1951 1952 - Revision history
2026-10-18T08:04:08Z
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Paul.xu at 23:17, 14 January 2014
2014-01-14T23:17:37Z
<p></p>
<p><b>New page</b></p><div>== Protein Expression ==<br />
<br />
2014/1/14<br> Paul <br />
<br />
[[Image:1951 1952Expression 1-2014.jpg]]<br> <br />
<br />
{| width="832" cellspacing="1" cellpadding="1" border="1"<br />
|-<br />
| Lane<br> <br />
| Name<br> <br />
| Protein Name<br> <br />
| Clone ID<br> <br />
| Host<br> <br />
| Induction<br> <br />
| Purification<br> <br />
| pI/Mw<br><br />
|-<br />
| 1<br> <br />
| pT7A3-HC-yFLP<br> <br />
| Tat-yFLP<br> <br />
| 1951<br> <br />
| <br />
5 ml,<br> <br />
<br />
NEB Turbo <br />
<br />
| 1 mM IPTG&nbsp; RT o/n<br> <br />
| <br />
Ni beads <br />
<br />
| <br />
9.76 / 52.8 <br />
<br />
|-<br />
| 2<br> <br />
| pT7A3-HC-yFLP<br> <br />
| Tat-yFLP<br> <br />
| 1951<br> <br />
| <br />
&nbsp;5 ml, <br />
<br />
NEB Turbo <br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| <br />
Ni beads <br />
<br />
| <br />
9.76 / 52.8<br> <br />
<br />
|-<br />
| 3<br> <br />
| pT7A3-HC-yFLP<br> <br />
| Tat-yFLP<br> <br />
| 1951<br> <br />
| <br />
5 ml, <br />
<br />
NEB Turbo <br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| <br />
Ni beads <br />
<br />
| <br />
9.76 / 52.8 <br />
<br />
|-<br />
| 4<br> <br />
| pT7A3-Tat-Flpo<br> <br />
| Tat-Flpo<br> <br />
| 1952<br> <br />
| <br />
5 ml, <br />
<br />
NEB Turbo <br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| <br />
Ni&nbsp; beads <br />
<br />
| <br />
9.76 / 52.8 <br />
<br />
|-<br />
| 6<br> <br />
| pT7A3-Tat-Flpo<br> <br />
| Tat-Flpo<br> <br />
| 1952<br> <br />
| 5 ml, <br />
NEB Turbo <br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| <br />
<br> <br />
<br />
Ni beads <br />
<br />
| <br />
9.76 / 52.8<br> <br />
<br />
|-<br />
| 7<br> <br />
| pT7A3-HC-yFLP<br> <br />
| Tat-yFLP<br> <br />
| 1951<br> <br />
| &nbsp; 5 ml, <br />
Nico21(DE3)pLysN <br />
<br />
<br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| <br />
Ni beads <br />
<br />
| <br />
9.76 / 52.8 <br />
<br />
|-<br />
| 8<br> <br />
| pT7A3-HC-yFLP<br> <br />
| Tat-yFLP<br> <br />
| 1951<br> <br />
| &nbsp;5 ml, <br />
Nico21(DE3)pLysN <br />
<br />
<br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| Ni beads<br> <br />
| <br />
9.76 / 52.8<br> <br />
<br />
|-<br />
| 9<br> <br />
| pT7A3-HC-yFLP<br> <br />
| Tat-yFLP<br> <br />
| 1951<br> <br />
| &nbsp; 5 ml, <br />
Nico21(DE3)pLysN <br />
<br />
<br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| <br />
Ni beads <br />
<br />
| <br />
9.76 / 52.8 <br />
<br />
<br> <br />
<br />
|-<br />
| 10<br> <br />
| pT7A3-Tat-Flpo<br> <br />
| Tat-Flpo <br />
| 1952<br> <br />
| 5 ml, <br />
Nico21(DE3)pLysN <br> <br />
<br />
| 1 mM IPTG RT o/n <br> <br />
| Ni beads <br> <br />
| 9.76 / 52.8 <br><br />
|-<br />
| 11<br> <br />
| Fermentas PageRuler Plus<br> <br />
| <br> <br />
| <br> <br />
| <br> <br />
| <br> <br />
| <br> <br />
| <br><br />
|-<br />
| 12<br> <br />
| purified T4 Ligase 2 ul of 38 mg/ml<br> <br />
| <br> <br />
| <br> <br />
| <br> <br />
| <br> <br />
| <br> <br />
| <br><br />
|}<br />
<br />
<br> <br />
<br />
Tat-yFLP has better expression than Tat-Flpo<br> <br />
<br />
Purification of Tat-yFLP on Ni column.<br><br />
<br />
[[Image:1951_pT7A3-HC-yFLP_purification.jpg]]<br><br />
<br />
Lysate from 8 L culture, Nico21(DE3)pLysN, 0.2 mM IPTG o/n. Load to 15 ml Ni column, <br />
<br />
Lane 1 first Ni column eluate and concentrated.<br><br />
<br />
Lane 2 and 4~12: Load 5X diluted protein solution to 2nd 5 ml GE pharmacia Ni column, elute by linear gradient of Imidazole. Fractions containing proteins<br />
<br />
Looks like protein expression is low, also not pure (lot of degradation)<br />
<br />
[[Category:Protein_Purification]]</div>
Paul.xu