protocol:ubiquitin

General Information

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

MCIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQCKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLCGG

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 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

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

Note: this protocol is adapted from OpenWetWare (if the link becomes invalid, please usethis 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.