I am performing azide-alkyne cycloaddition using a propargylated small molecule (alkyne) which is getting incorporated into proteins and biotin azide for pulldown by strepavidin beads. I am seeing non-specific binding in all of my samples and I am unsure where the problem is occuring. When I perform this reaction on fixed cells I get very clean results, with signal in my treated groups, and nothing in my vehicle controls. However in lysates I get identical bands in every group when I run the purified samples on a gel. Please help!
I am using this protocol https://vectorlabs.com/productattachments/protocol/Cell-Lysata-Labeling-rev2.pdf
For each azide or alkyne- modified protein lysate sample, add the following to a 1.5 mL microfuge tube, then vortex briefly to mix. 50 µL protein lysate (1-5 mg/mL) in protein extraction buffer 100 µL PBS buffer 4 µL corresponding azide or alkyne detection reagent (20 μM final concentration)Add 10 µL of 100 mM THPTA solution, vortex briefly to mix. Add 10 µL of 20 mM CuSO4 solution, vortex briefly to mix. Add 10 µL of 300 mM sodium ascorbate solution to initiate click reaction, vortex briefly to mix. Protect reaction from light and allow click reaction to incubate for 30 minutes at room temperature. Longer reaction time might improve labeling efficiency.
https://help.lumiprobe.com/p/45/nonspecific-protein-labeling-click-chemistry
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