TITLE: Purification of (Kai) proteins via size exclusion chromatography
AUTHORS: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to further purifiy (Kai) proteins via size exclusion chromatography using either HiPrep 16/60 Sephacryl S-300 HR column (120 ml column volume), HiPrep 16/60 Sephacryl S-200 HR (120 ml column volume) or Superdex 200 Increase 10/300 GL (24 ml column volume)</div></div>

[STEPS]
?. [preparation of buffer and solutions]
Prepare 1 l of each: Degassed MiliQDegassed 20 % EtOHDegassed running buffer [20-50 mM Tris/HCl (pH8), 150 mM NaCl, 1-2 mM DTT, only for KaiC proteins: 5 mM MgCl2, 1 mM ATP]
?. [set-up of your liquid chromatography system]
Connect degassed MilliQ with pump A and pump B of your chromatography instrument
?. [set-up of your liquid chromatography system]
Purge and rinse all valves and sample loop with MilliQ (pump A and pump B)
?. [set-up of your liquid chromatography system]
Connect an appropriate sample loop (e.g. 2 ml) to your system and rinse with MilliQ
?. [set-up of your liquid chromatography system]
Connect a size exclusion chromatography column to your system (e.g. Superdex 200 Increase 10/300 GL, HiPrep 16/60 Sephacryl S-200 HR, HiPrep 16/60 Sephacryl S-300 HR column)
?. [set-up of your liquid chromatography system]
Wash column with at least 0.5 column volumes degassed MilliQNote: make sure not to exceed the maximal pressure the column can withstand. Recommended maximal flow rates: for sephacryl S-200 and sephacryl S-300: 0.5 ml/minfor superdex 200:  0.75 ml/min
?. [set-up of your liquid chromatography system]
Connect running buffer as eluant A and purge
?. [equilibration of the column]
Equilibrate the column with at least 1.5 column volumes buffer ANote: make sure not to exceed the maximal pressure the column can withstand. Recommended maximal flow rates: for sephacryl S-200 and sephacryl S-300: 1 ml/minfor superdex 200:  0.75 ml/min
?. [protein separation]
Remove aggregates and precipitates in your protein sample by centrifugation or filtration (use a syringe filter)Apply your protein to the sample loop (injection valve must be set to load position).
?. [protein separation]
Separate in 1 column volume running buffer using the following flow rates:for sephacryl S-200 and sephacryl S-300: 0.4-0.5 ml/minfor superdex 200: 0.75 ml/min
?. [protein separation]
Shortly before void proteins will be eluted: start to collect fractions of 0.5 ml – 1 ml
?. [protein separation]
Note: to estimate the size of (the oligomeric states of) your proteins, separate a standard solution (e.g. Biorad gel filtration standard) under the same conditions
?. [cleaning/storage]
Wash with at least 1.5 column volumes MilliQ (pump B)Note: make sure not to exceed the maximal pressure the column can withstand. Recommended maximal flow rates:for sephacryl S-200 and sephacryl S-300: 0.4-0.5 ml/minfor superdex 200: 0.75 ml/minAlternatively: wash with 1 column volume buffer, rinse with 0.5 column volumes MilliQ and equilibrate with 2 column volumes buffer for the next separation (in this case you can skip cleaning with EtOH described in steps 14 and 15)
?. [cleaning/storage]
Connect pump A to 20 % EtOH and purge
?. [cleaning/storage]
Wash with at least 1.5 column volumes 20 % EtOH (pump A)Note: make sure not to exceed the maximal pressure the column can withstand. Recommended maximal flow rates:for sephacryl S-200 and sephacryl S-300: 0.2 ml/minfor superdex 200: 0.4 ml/min
?. [qualitative analysis of eluted fractions]
Choose fractions of interest based on the absorption at 280 nm
?. [qualitative analysis of eluted fractions]
For each fraction of interest, pipette 80 µl of Bradford solution in a well of a 96 well plate and add 5-20 µl of your fraction. Colour change to blue indicates that you successfully eluted proteins. Keep those fractions
?. [qualitative analysis of eluted fractions]
Control homogeneity and size of your eluted protein(s) by separation via SDS-PAGE
?. [qualitative analysis of eluted fractions]
Measure protein concentration in the fraction of interest (using e.g. Bradford method or infrared spectrometer (direct detect instrument, Merck))Note: If necessary, you can concentrate your protein using a disposable centrifugal concentrator