Haynes Lab:Notebook/Engineering PC-TFs/2014/09/25: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2014/09/25 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
(fix raw html notebook nav)
 
(One intermediate revision by one other user not shown)
Line 2: Line 2:
|-
|-
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
|style="background-color: #800000" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #800000" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Summary==
==Summary==
*  
* Purified PcTF protein.
----
'''Zymo-Spin P1 Column His-tag Purification Kit'''<br>
Protein kept on ice during procedure; centrifuge in cold room used.<br>
*Vortexed His-Affinity Gel briefly.
*Transferred 250μL of His-Affinity Gel to the Zymo-Spin P1 column.
*Placed column in 2mL collection tube.
*Centrifuged for 20 seconds at max rpm.
*Discarded flow-through.
*Added 300μL PcTF in protein extraction buffer to column.
*End-over-end mixing for two minutes to resuspend gel and allow protein to bind to resin.
*Centrifuged for 20 seconds at max rpm.
*Discarded flow-through.
*Added 250μL His-Wash Buffer and mixed to resuspend gel.
*Centrifuged for 20 seconds at max rpm.
*Discarded flow-through.
*Repeated wash step once.
*Transferred column into a labeled 1.7mL microcentrifuge tube.
*Added 150μL His-Elution BUffer and resuspended gel via end-over-end mixing for one minute.
*Centrifuged for 20 seconds at max rpm.
*Discarded column and stored purified protein at -20°C.




Latest revision as of 00:21, 27 September 2017



 		Main project page
Previous entry      Next entry

Summary

  • Purified PcTF protein.

Zymo-Spin P1 Column His-tag Purification Kit
Protein kept on ice during procedure; centrifuge in cold room used.

  • Vortexed His-Affinity Gel briefly.
  • Transferred 250μL of His-Affinity Gel to the Zymo-Spin P1 column.
  • Placed column in 2mL collection tube.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Added 300μL PcTF in protein extraction buffer to column.
  • End-over-end mixing for two minutes to resuspend gel and allow protein to bind to resin.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Added 250μL His-Wash Buffer and mixed to resuspend gel.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Repeated wash step once.
  • Transferred column into a labeled 1.7mL microcentrifuge tube.
  • Added 150μL His-Elution BUffer and resuspended gel via end-over-end mixing for one minute.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded column and stored purified protein at -20°C.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2014/09/25&oldid=1015454"