User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/25: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" 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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==Procedure== | ||
#Prepare a 300uM solution of sodium dithionite in 100mL of Tris buffer at pH 8.3 | |||
#Degas solution for an hour | |||
##Nitrogen was bubbled through the solution | |||
#A disposable column was obtained | |||
##GE PD-10 pre-packed column | |||
#The column was cleaned by running 25mL of dionized water through it | |||
#The column was equilibrated by running 25mL of the degassed solution through it | |||
#Once equilibrated, we started running the protein in the following steps | |||
##2.5mL of protein are added to the column (do not collect elute) | |||
##3.5mL of degassed Tris with DTT are added to the column (collect elute) | |||
##25mL of degassed Tris with DTT are added to the column to re-equilibrate (do not collect elute) | |||
##Repeat the steps until all the protein has been run through the column | |||
#After the first collection, a solution of Ruthenium was added to the elute and the following collations (of protein) were added to the same tube | |||
##The ruthenium solution was made up to contain 10X more Ruthenium than protein | |||
#Once all the protein were collected from the column, the tube was covered in foil and placed on the shaker for 4 hours | |||
#During the wait period, the desalting column on the FPLC was cleaned and equilibrated with Tris buffer pH 8.3 | |||
#After 4 hours of reactions, the protein solution was run through the desalting column | |||
#The protein and ruthenium excess were separated using the column and stored in separate tubes | |||
#The tubes were placed in the freezer until further purification next week | |||
==To do next week== | |||
#Concentrate protein from desalting column | |||
#Clean and restabilize desalt column | |||
#Run through Q-column w/ gradient 0-30% NaCl 10 minutes and collect fractions | |||
#concentrate elute from q-column | |||
#Run through Q-column w/ gradient 0-30% NaCl 30 minutes and collect fractions | |||
#concentrate elute from q-column | |||
#Run through Q-column w/ gradient 0-30% NaCl 1 hour and collect fractions | |||
#concentrate elute from q-column | |||
#run gel | |||
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Latest revision as of 00:08, 27 September 2017
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Procedure
To do next week
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