Jul 11, 2019

Public workspacePurification of (Kai) proteins via size exclusion chromatography

Journal of Bacteriology
DOI
dx.doi.org/10.17504/protocols.io.mdtc26n
  • 1Heinrich-Heine Universität Düsseldorf;
  • 2Institute of Biology III, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany;
  • 3Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
  • 4Institute for Microbiology and Molecular Biology, Justus-Liebig University, 35392 Giessen, Germany;
  • 5College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
  • 6Institute for Synthetic Microbiology, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University Duesseldorf, 40225 Duesseldorf, Germany
  • Axmann Lab
  • CyanoWorld
Anika Wiegard
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DOI: dx.doi.org/10.17504/protocols.io.mdtc26n
External link: https://doi.org/10.1128/JB.00478-19
Protocol Citation: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria IM Axmann 2019. Purification of (Kai) proteins via size exclusion chromatography. protocols.io https://dx.doi.org/10.17504/protocols.io.mdtc26n
Manuscript citation:
Wiegard A, Köbler C, Oyama K, Dörrich AK, Azai C, Terauchi K, Wilde A, Axmann IM, Array. Journal of Bacteriology 202(4). doi: 10.1128/JB.00478-19
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 21, 2017
Last Modified: July 11, 2019
Protocol Integer ID: 9363
Keywords: Kai proteins, SEC, size exclusion chromatography, gel filtration, superdex 200, sephacryl S200, sephacryl S300 FPLC, protein purification
Abstract
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)
Guidelines
Choose the chromatography column depending on the amount and size of your protein. Purification can be performed at 4 °C or 30 °C depending on the stability of your protein.
Materials
MATERIALS
ReagentMilliQ water
ReagentMagnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)
ReagentTris(hydroxymethyl)aminomethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #252859-500G
ReagentNaCl
ReagentHiPrep Sephacryl S-200 HR ColumnGE HealthcareCatalog #17116601
ReagentHiPrep Sephacryl S-300 HR ColumnGE HealthcareCatalog #17116701
ReagentSuperdex 200 Increase 10/300 GL ColumnGE HealthcareCatalog #28990944
ReagentEtOH
ReagentHCl
Reagent14 Dithiotreitol (DTT)Carl RothCatalog #6908.1
ReagentAdenosin-5-triphosphate disodium salt (ATP)Carl RothCatalog #HN35.1
ReagentQuick Start™ Bradford 1x Dye Reagent Bio-Rad LaboratoriesCatalog #5000205
STEP MATERIALS
ReagentGel Filtration StandardBio-Rad LaboratoriesCatalog ##1511901
You will further need:
  • reaction tubes
  • 96 well plate
  • centrifugal concentrators with appropriate MWCO
  • chromatography system
Protocol materials
ReagentQuick Start™ Bradford 1x Dye Reagent Bio-Rad LaboratoriesCatalog #5000205
ReagentHiPrep Sephacryl S-200 HR ColumnGE HealthcareCatalog #17116601
ReagentSuperdex 200 Increase 10/300 GL ColumnGE HealthcareCatalog #28990944
ReagentMilliQ water
ReagentAdenosin-5-triphosphate disodium salt (ATP)Carl RothCatalog #HN35.1
ReagentGel Filtration StandardBio-Rad LaboratoriesCatalog ##1511901
ReagentTris(hydroxymethyl)aminomethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #252859-500G
ReagentNaCl
ReagentHiPrep Sephacryl S-300 HR ColumnGE HealthcareCatalog #17116701
ReagentEtOH
ReagentHCl
ReagentMagnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)
Reagent14 Dithiotreitol (DTT)Carl RothCatalog #6908.1
ReagentGel Filtration StandardBio-Rad LaboratoriesCatalog ##1511901
preparation of buffer and solutions
preparation of buffer and solutions
Prepare 1 l of each: 
  • Degassed MiliQ
  • Degassed 20 % EtOH
  • Degassed 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
set-up of your liquid chromatography system
Connect degassed MilliQ with pump A and pump B of your chromatography instrument
Purge and rinse all valves and sample loop with MilliQ (pump A and pump B)
Connect an appropriate sample loop (e.g. 2 ml) to your system and rinse with MilliQ
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)
Wash column with at least 0.5 column volumes degassed MilliQ
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: 0.5 ml/min
  • for superdex 200:  0.75 ml/min
Connect running buffer as eluant A and purge
equilibration of the column
equilibration of the column
Equilibrate the column with at least 1.5 column volumes buffer 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: 1 ml/min
  • for superdex 200:  0.75 ml/min
protein separation
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).
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/min
  • for superdex 200: 0.75 ml/min
Shortly before void proteins will be eluted: start to collect fractions of 0.5 ml – 1 ml
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
ReagentGel Filtration StandardBio-Rad LaboratoriesCatalog ##1511901
cleaning/storage
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/min
  • for superdex 200: 0.75 ml/min
Alternatively: 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)
Connect pump A to 20 % EtOH and purge
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/min
for superdex 200: 0.4 ml/min
qualitative analysis of eluted fractions
qualitative analysis of eluted fractions
Choose fractions of interest based on the absorption at 280 nm
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
Control homogeneity and size of your eluted protein(s) by separation via SDS-PAGE
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