Sep 23, 2024
Size Exclusion Chromatography
- Verena Dederer1,2,3,
- Stefan Knapp1,2,3,
- Sebastian Mathea1,2,3
- 1Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, Frankfurt 60438, Germany;
- 2Structural Genomics Consortium, Buchman Institute for Molecular Life Science (BMLS), Max-von-Laue-Straße 15, Frankfurt 60438, Germany;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Verena Dederer, Stefan Knapp, Sebastian Mathea 2024. Size Exclusion Chromatography. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r55ng1b/v1
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: September 05, 2024
Last Modified: September 23, 2024
Protocol Integer ID: 106977
Keywords: separating protein, smaller protein, size exclusion chromatography size, protein size, proportional to the protein size, protein sample, exclusion chromatography, size separation, protein, small molecule, large molecule, solvent, packed with porous bead, porous bead, stable interaction
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000519
Abstract
Size-exclusion chromatography (SEC) is a method for separating proteins according to their size. To achieve this, a protein sample is applied to a column that is tightly packed with porous beads.
The size separation is determined by the thickness of the hydration shell of the proteins in a solvent. Small molecules can penetrate the pores, while large molecules cannot and are eluted first from the column. Smaller proteins follow, whereby the elution time is inversely proportional to the protein size.
We used this method to investigate whether proteins form a stable complex. The individual complex partners have a characteristic elution profile. If there is a stable interaction, they are expected to co-elute after mixing.
Troubleshooting
Sample Preparation
Mix the two purified interacting proteins in appropriate buffer with the appropriate ratio.
In our case: Protein 2 LRRK2RCKW + Protein 2 DARPin E11 with a ratio of 1:5 in 2 mL buffer
Note
Buffer can be any buffer your proteins of interest behave well.
Sample Analysis
2 mL sample were subjected to a S200 gel filtration column combined with an ÄKTA XPress system in running buffer: 20 mM HEPES pH 7.4, 500 mM NaCl, 2.5 mM MgCl2, 20 µM GDP, 0.5 mM TCEP, 0.5%
glycerol
Collect flowthrough fractions (e.g. 3-mL)
Analyse collected fractions by SDS PAGE and Coomassie stain or immunoblot analysis.