I would like to purify and check the oligomerization states of my protein using Size Exclusion Chromatography (SEC). The molecular weight of my protein is ~60 kDa and it easily aggregates during concentration. I know during SEC I need to concentrate the protein before loading in the column. Could anybody please suggest me is there any suitable SEC column which loads around 2 to 5 ml sample and shows high resolution during small scale purification?
Dear Sir. Concerning your issue about the suitable SEC column for purification of the protein. In recent years, the use and number of biotherapeutics has increased significantly. For these largely protein-based therapies, the quantitation of aggregates is of particular concern given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). In the following review we will outline the history and background of SEC for the analysis of proteins. We will also discuss the instrumentation for these analyses, including the use of different types of detectors. Method development for protein analysis by SEC will also be outlined, including the effect of mobile phase and column parameters (column length, pore size). We will also review some of the applications of this mode of separation that are of particular importance to protein biopharmaceutical development and highlight some considerations in their implementation.I think the following below links may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556795/pdf/ljlc35_2923.pdf
https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=19069
Thanks
Dear Mahfuza
I think that condidering the size of your monomeric protein that is similar to bsa the supedex 200 coloumn is a good candidate for separate monomer from dimers and aggregates.
2 to 5ml could be loaded in the 26/60 coloumn (coloum volume 330ml) but of course it is a semipreparative coloumn suotable for separationof protein from aggeegtes and recovery of the monomeric protein but do you need to load on it some protein mg.
Other available formats: 16/60 (coloumn volume 120ml) you can load up to 1.5ml
10/30 supedex 200 increase (coloum volume 24ml) --> higher resolution but you cal load 100-200ul)
This prepacked coloumns are quite expensive (1000-2000 euro) bua can be washed and re-used for long time.
Both 26/60 and 16/60 coloumn need about 2h for a single run. 10/30 around 1h at the suggested flow rates.
Good luck
Manuele
Hi Mahfuza - Here is a loading capacity for our 3um silica SEC-HPLC columns for typical QC standards.
- http://www.sepax-tech.com/app_note_access.php?id=SM1003
For (2 to 5 ml sample) separations, If you are looking to cut down your run times, you could also try our SRT10/10C product line:
- http://www.sepax-tech.com/app_note_access.php?id=SP1019
- http://www.sepax-tech.com/app_note_access.php?id=SP1005
We even scale up to 5 cm ID columns;
- http://www.sepax-tech.com/app_note_access.php?id=SP1022
Salt in the mobile phase can potentially play a role on the oligomerization state of the protein and artifacts, for example, some people add 150-300 mM Na2SO4 to a standard phosphate running buffer.
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