Difference between revisions of "Team:Freiburg/Protocols/Purification"
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− | <div class="tags"><span> | + | <div class="table sectionedit2"><table class="inline"> |
+ | <tr class="row0"> | ||
+ | <th class="col0" colspan="2"> <strong>buffers</strong> </th> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0"> <strong>standard buffer/lysis buffer</strong> </td><td class="col1"> 20 mM Tris pH 7.5 </td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0"> </td><td class="col1"> 150 mM NaCl </td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0"> </td><td class="col1"> 0.3 mM PMSF </td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0"> </td><td class="col1"> 10mM Imidazole </td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0"> <strong>washing buffer</strong> </td><td class="col1"> 20 mM Tris pH 7.5 </td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0"> </td><td class="col1"> 150 mM NaCl </td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0"> </td><td class="col1"> 20 mM Imidazole </td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0"> <strong>elution buffer</strong> </td><td class="col1"> 20 mM Tris pH 7.5 </td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0"> </td><td class="col1"> 150 mM NaCl </td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0"> </td><td class="col1"> 500 mM Imidazole </td> | ||
+ | </tr> | ||
+ | </table></div> | ||
+ | <!-- EDIT2 TABLE [1727-2024] --><div class="tags"><span> | ||
<a class="wikilink1" href="/igem2015/doku.php?id=tag:protocols&do=showtag&tag=protocols" rel="tag" title="tag:protocols">protocols</a>, | <a class="wikilink1" href="/igem2015/doku.php?id=tag:protocols&do=showtag&tag=protocols" rel="tag" title="tag:protocols">protocols</a>, | ||
<a class="wikilink1" href="/igem2015/doku.php?id=tag:protpur&do=showtag&tag=protpur" rel="tag" title="tag:protpur">protpur</a> | <a class="wikilink1" href="/igem2015/doku.php?id=tag:protpur&do=showtag&tag=protpur" rel="tag" title="tag:protpur">protpur</a> |
Revision as of 13:17, 26 August 2015
Protein Purification on gravity flow columns
improved protocol for classical gravity flow columns
material:Gravity flow columns, 50% Ni-NTA agarose in storage buffer, ddH<sub<2</sub>O, Lysis buffer
duration:
All buffers have to be degassed before by applying sonfication for example
To increase the flow though velocity the collection tubes (falcon tubes with cap) were connected to a vacuum pump with interposed pressure regulation. beware of applying too much pull to avoid ait bubble formation near the diaphragma.
column packing
- wash columns with 5 mL of aqua dest
- close columns with plug
- slowly pipette 1 mL of 1:1 NTA-agarose/stoarge buffer slurry on the diaphragma
- let the agarose settle out
- open the tube and wash the agarose with 2-3 mL lysis buffer
loading
- change the falcon tube to elute the sample in
- pipette the cell lysate onto the column and let almost flow through
- collect flow-through and change collection tube again
- wash with 5 mL washing buffer and collect FT
- change collection tube
- elute 5 times with 500 µL of elution buffer
- every time the liquid surface is near the bed: add new buffer and collect the flow though that accumulated until then
washing
- add 500µL - 1mL of total elution buffer and let flow through
- wash with 3 mL of aqua dest to remove imidazol
- add 1 mL of 20 % ethanol and let flow though until around 0.5 mL left
- close column with plug and store @ 4°C
reusing
- equilibrate the column with lysis buffer as described above
- use one column only for one type of protein!
buffers | |
---|---|
standard buffer/lysis buffer | 20 mM Tris pH 7.5 |
150 mM NaCl | |
0.3 mM PMSF | |
10mM Imidazole | |
washing buffer | 20 mM Tris pH 7.5 |
150 mM NaCl | |
20 mM Imidazole | |
elution buffer | 20 mM Tris pH 7.5 |
150 mM NaCl | |
500 mM Imidazole |