Hi all,
I am considering using microscale thermophoresis to characterise the binding of oligosaccharides (700 Da) to a protein (~ 30 kDa). I realise in this case I could label the protein, but I may end up wanting to use many different mutants and this would involve the labelling of all of them. So, I am wondering how practical an alternative it would be to label the oligosaccharide(s).
I am unable to source directly fluorescently-labelled oligosaccharides, but I am able to source them in a biotinylated form. So, my question would be, could I pre-form a complex between these biotinylated oligosaccharides and fluorescently labelled streptavidin, and then use these in MST with a titration of the protein? Another question might be how to separate streptavidin:oligosaccharide complexes from any free streptavidin - maybe use a biotin resin to bind the excess?
Many thanks for any advice.
Hi Roger,
In principle your idea using biotinylated oligosaccharides and fluorescently labeled streptavidin should work. However, labeled streptavidin as secondary label is difficult since it is a tetramer and you can very easily produce very large multimers / aggregates. Therefore, I would recomment do either use a large excess of biotinylated oligosaccaride over streptavidin, or a biotinylation ratio
Hi Heide,
Many thanks for the advice. I hadn't considered the tetrameric nature of SA. If I do go ahead with this approach, I will use a large excess of oligosaccharide as you suggest.
Thanks,
Roger
Hi Heidi,
If you have reducing saccharides you can add a fluorescent label to the reducing end. See:
Jackson,P. (1990) Biochem. J. Vol 270, p705
Jackson, P. (1994) Methods Enzymol. Vol230, 9250
You can also do a search for FACE+ saccharide
Thanks Peter!
I guess this is an easy approach for chemistry labs, but I'm afraid that not every biochemistry lab would be able to do it. But definitely good to know. Thanks again!
The labelling protocol is not difficult and requires standard biochemical bench equipment; not a chemistry lab. It is done on a micro scale ( microlitres) and the sensitivity on a PAGE gel is in the low picomole level. Gels can be viewed on a UV light box. It is done almost daily in the Dept of Biochemistry in the University of Cambridge UK ( Dupree lab) by biologists and bichemists with no major qualifications in chemistry.
Labeling glycans is extremely simple with 2-aminobenzoic acid (2-AA, anthranilic acid, AA).
Reference: K. R. Anumula. (2014) Single Tag for Total Carbohydrate Analysis. Anal. Biochem. 457, 31-37.
Maybe a labelfree MST approach would be an elegant alternative. Of course only if your protein contains tryptophane (s) and if you have access to a Monolith labelfree.
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