===== General Information =====
  * WT:
<code>
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
</code>
  * Q2C:
<code>
MCIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
</code>
  * D32C:
<code>
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQCKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
</code>
  * R74C:
<code>
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLCGG
</code>
  * Note: for R74C, the protein cannot be caught by SPFF due to the lower PI (see table below). My solution is to collect flow-through. Maybe it is worth trying QFF column (may or may not work).

|Variants|Molecular Weight|PI|charge at pH5.2|Extinction Coefficient at 280nm|
|WT|8564.8203|7.19|2.5|1280 M-1cm-1|
|Q2C|8539.8291|7.19|2.5|1280 M-1cm-1|
|D32C|8552.8711|8.17|3.3|1280 M-1cm-1|
|R74C|8511.7725|6.06|1.5|1280 M-1cm-1|

===== Buffer =====
  * Buffer A: sodium acetate 50mM, pH=5.2
  * Buffer B: sodium acetate 50mM, NaCl 1M, EDTA 1mM, pH=5.2
  * Buffer C: sodium acetate 50mM, NaCl 150mM, EDTA 1mM, pH=5.2
  * Produre
    * 2.9L ddH2O; add NaAc 9.72g; add HAc until pH=5.2
    * separate into three part
<code>
Part 1: 1L ==> Buffer A
Part 2: 1L + 58.44g NaCl + 0.372g EDTA ==> Buffer B
Part 3: 1L + 8.76g NaCl + 0.372g EDTA ==> Buffer C
</code>


===== Procedure =====
  * Note: this protocol is adapted from [[http://openwetware.org/wiki/Purifying_Ubiquitin_from_expression_lysates | OpenWetWare]] (if the link becomes invalid, please use{{:protocol:openwetware.pdf|this pdf file}}
  * Resuspend E. coli in 20mL Buffer A; French press 2~3 times (3 times if using Low's old machine).
  * Centrifuge @18000rpm,4C for 30 min; Transfer supernatant to a culture tube (always keep it in ice from here on)
  * Adjust PH by acetic acid to 4.5~5.0 and the solution will become milky; Centrifuge @18000rpm,4C for 10 min.
  * Transfer supernatant back to the culture tube; Adjust PH to 5.2
  * Filter the solution using filter syringe.
  * Run the sample through SPFF column (cation exchange column; Pharmacia Fast Flow SP resin ).
  * Run appropriate fractions through Gel-filtration column.
===== Reference =====
  * {{:protocol:ubq_purify.pdf|pdf}}