My aim is to detect nascent protein synthesis by click-chemistry of L-homopropargyl-glycine-labelled proteins with biotin azide and detection by western blotting with fluorescently-conjugated streptavidin. So far, I have failed to detect labelling above what looks like background biotinylation. I'm using biotin azide from Biotium (Cat# 92167), but I've noticed there different biotin azide molecules with different molecular weights. I was wondering if there were differences in their applicability for copper-catalysed click-chemistry. Any help with troubleshooting this would be appreciated. Cheers!
There are several biotin-azide substrates available for click-chemistry, and their selection depends on factors such as their solubility, reactivity, stability, and specificity. Biotin azide (Cat# 92167) from Biotium is a popular choice and has been widely used in the literature for labeling nascent proteins in cells. However, it is possible that the labeling efficiency may vary depending on the experimental conditions and the nature of the sample.
One option you could consider is to try different biotin-azide substrates to see if there is any improvement in labeling efficiency. For example, some other commercially available biotin-azide substrates include dibenzocyclooctyne (DBCO)-biotin azide and cyclooctyne-biotin azide. These substrates have different molecular weights and structures, and their reactivity may differ from that of Biotium's biotin azide.
Another option to consider is the copper-catalyst used for the click-chemistry reaction. Some copper-catalysts may be more efficient for particular substrates or reaction conditions. You could try different copper-catalysts or adjust the reaction conditions to optimize the click-chemistry efficiency.
Overall, the best biotin-azide substrate for your experiment would depend on the specific conditions and requirements of your study. It may be helpful to consult with experts in the field or refer to relevant literature to help guide your selection.
Dear @Jonas,
The spacer length should not affect reactivity. These molecules rather differ in the distance between biotin and the reactive end. However, for a good interaction between biotin and streptavidin, you need a certain minimum spacer length, as the biotin needs to fit into one of the pockets of the streptavidin tetramer. To this end, I'd recommend at least an added C6 chain, like it is the case in biotin-X-Osu, or 2 or more PEG monomers. So better make sure the biotin-azide you are referring to has this spacer.
If your reaction is taking place in a biochemically active system, I'd strongly recommend to use copper free click chemistry instead of the azide/alkyne system, like DABCO (as Satyendra Singh already suggested), and a suitable diene like e.g., a tetrazine.
This brochure by Iris Biotech might give you some ideas on biocompatible click chemistry: https://media.iris-biotech.de/flyers/IF4_1_Click_Chemistry.pdf, there are some DBCO and Tz labeled molecules which might help you succeed.
A nice chart with some more exotic click structure is in this paper: TMTHSI, a superior 7-membered ring alkyne containing reagent for strain-promoted azide-alkyne cycloaddition reactions; J. Weterings, C. J. F. Rijcken, H. Veldhuis, T. Meulemans, D. Hadavi, M. Timmers, M. Honing, H. Ippel, R. M. J. Liskamp; Chem. Sci. 2020; 11: 9011-9016. https://doi.org/10.1039/d0sc03477k
HTH,
Wolfgang
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