Using a MALS system together with size exclusion chromatography (SEC)? Not performing as expected? Tap into these column tips for how to solve this and other issues.
Measuring mass of macromolecules
Size exclusion chromatography (SEC) separates molecules based on hydrodynamic volume. If you need to identify the molecules, multi-angle static light scattering (MALS) is a common technique to use in combination with SEC.
MALS is commonly used in-line in a combined SEC-MALS setup. The weight average molecular mass is determined for molecules passing a detector cell. The principle is based on reading the intensity and the angular dependence of the scattered light.
SEC column tips for great MALS results
MALS is a very precise measurement, but difficulties might arise when working with a SEC-MALS setup. I have listed four possible issues and tips for how you can overcome these.
- Problem: high mass species knocked off the column by the injection itself (by a pressure pulse, by detergents in the buffer, etc.
To diagnose: perform a blank run prior to running samples. Tip: do not increase the flow too rapidly.
- Problem: nonspecific binding to the SEC resin resulting in the proteins eluting later than expected.
Tip: screen buffer conditions.
- Problem: high mass species co-elutes with the target mass. This results in a much higher mass than expected.
Tip: screen buffer conditions.
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Problem: difficulties detecting components present in small quantities.The concentration is just too low to get good MALS data.
Tip: increase separation efficiency and concentrate.
I also recommend having a dedicated SEC-MALS setup for molecular mass determinations. This will ensure an effective station for identification of target molecules. Never turn the flow off. A system that runs on a low flow (re-circulation) does not suffer from pressure-induced signals in the SEC-MALS setup.
To get a good baseline, you should wash the column before the first injection with several column volumes of water and buffer (often overnight with a low flow rate). Do not use the SEC column at the maximum flow rate, though.
Recent SEC-MALS applications
Interested in recent applications where GE’s Superdex 200 Increase 10/300 GL columns have been used for SEC-MALS? Start reading here:
- Heintz, U., and Schlichting, I. Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence. eLife. 2016; 5: e11860 [Online]. (12 January 2016).
- Gaik, M. et al. Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold. J Cell Biol. 208(3), 283–297 (2015)
- Thong, S. et al. Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry. eLife 2016;5:e19042 [Online]. (16 August 2016).
- Yan, R. et al. The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS. PLoS One. 2016; 11(7): e0157895 [Online]. (18 July 2016).