- This topic has 6 replies, 6 voices, and was last updated 1 month ago by Mikael Andersson.
January 25, 2024 at 14:13 #5061
We are seeing a slightly higher elution volume than expected and a tailing in SEC profiles of a glycoprotein in a complex mixture of other proteins. We are monitoring the elution of this specific glycoprotein in the fractions with an antibody. Wondering if this is due to glycosilation heterogeinity but before going further chasing this hypoteses I would like to rule out non specific binding to the superdex 200 matrix. Does anyone have any experience with binding of carbohydrates to this kind of matrixes and of ways of counteract them?
January 25, 2024 at 14:24 #5062
I have seen non specific interactions with the matrix mainly for HPLC type columns with silica.
For superdex type resins not so much, but very possible. But I also notice for some proteins there is tailing. Also to consider would be different dynamic conformations that have different hydrodynamic radii
January 25, 2024 at 14:45 #5063
At the VIB Protein Core we also see this regularly for glycosylated proteins on Superdex 200 columns. The elution volume can become quite a bit higher than expected (especially for larger sugar structures). We get the confirmation on molecular weight by checking on SEC-MALLS and performing glycosylation analysis.
January 25, 2024 at 15:56 #5064
As others have already commented we also get tailing/retention on Superdex pg media with glycosylated proteins.
We found that increasing the NaCl always helped reduce this effect for SEC (or desalting) of glycosylated proteins. For glycosylated proteins we routinely use PBS+300mM NaCl for DS/SEC so it’s still pretty much compatible with downstream tissue culture work.
Hope that helps.
- This reply was modified 1 month ago by Nick Berrow.
January 26, 2024 at 10:09 #5066
I had this problem once with human FactorXa protein on a Superdex 200 SEC column. It was a commercial batch that we had paid 10k€ for, so I started sweating when the protein didn’t show up at the expected volume. It eventually eluted with some delay. Higher salt concentrations could help reducing the interations, if your protein tolerates that.
January 26, 2024 at 11:00 #5067
Thank you all.
I will try the 300 mM salt and see out it plays out.
Thank you again.
January 26, 2024 at 11:09 #5068
We basically only produce produce glycosylated proteins in our facilty and as others here have indicated they all produce wide peaks. When we have done SEC-MALS on the different fractions of the peaks it is clear that they corresponds to differet sizes too. This size difference of peak subfractions is also something that we have seen in SDS page too. We thus attribute it to the heterogeneous glycosylation as you say.
Another thing to think of is that several purifications can sort proteins on glycosylations so if the glycan profile is of importance downstream you can keep that in mind when you work with glycosylated proteins and select your fractions etc.
- This reply was modified 1 month ago by Mikael Andersson.
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