2.4. Click chemistry

First, 2.5-mg protein of LV from heavy AHA (H-AHA)-labeled ISO mice or sham mice and 2.5-mg protein of LV from light AHA (L-AHA)-labeled sham mice or ISO mice were mixed together as one biological replicate. For each biological replicate, the H-AHA/L-AHA mixture was divided into twenty aliquots (0.25 mg/aliquot). A click reaction was performed on each aliquot as previously published (Speers and Cravatt 2009). Briefly, for each click reaction, the following reagents were added in this order: 1) 30 ul of 1.7-mM TBTA, 2) 8 ul of 50-mM Copper Sulfate, 3) 8 ul of 5-mM Biotin-Alkyne (C₂₁H₃₅N₃O₆S, Click Chemistry Tools), and 4) 8 ul of 50-mM TCEP. PBS was then added to a final volume of 400 ul and incubated for 1 hour at room temperature. Methanol/Chloroform precipitation was performed, and the precipitated protein was combined so that there would only be one pellet per 5-mg starting material.