I am labelling myristoylated proteins in cells and performing a click chemistry reaction to add the fluorescent TAMRA-alkyne to visualise via in-gel fluorescence. In a negative control sample containing no myristic acid label I am still seeing fluorescent bands similar to my samples containing the label. Does anyone know how to resolve this? Is there a way to block lysed protein prior to the click reaction?
The protocol is briefly explained below:
1. Label cells with myristic acid (azide) label https://www.thermofisher.com/order/catalog/product/C10268#/C10268
2. Lyse cells according to the Click IT protein reaction buffer kit protocol
https://www.thermofisher.com/order/catalog/product/C10276?uk&en#/C10276?uk&en
3. Add TAMRA alkyne and perform click reaction according to the kit protocol, precipitate proteins as per protocol, resuspend protein pellet in 30ul LSB + DTT and heat 70c for 10 mins prior to loading on gel
https://www.thermofisher.com/order/catalog/product/T10183?uk&en#/T10183?uk&en
Sorry I don't know. I find click reaction where the biomolecule is an azide and the detection moiety is the alkyne dye tends to have non-specific side reaction. I don't know why that is. You could possibly use a copper free reaction using one the strained ring alkyne dyes (DIBO-dye) that I would expect to have fewer side reactions. It is a slow reaction, usually left to react overnight. You might also call up Thermo Scientific Tech Assist and as for Courtenay Kerndt who did much of the click gel work in the early days of developing the click PTMs.
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