In-Gel Fluorescence to Monitor Protein Labeling

After labeling with Aha, aerial tissues were immediately harvested, flash frozen with liquid nitrogen, and stored at −80°C until subsequent workup steps. Frozen tissues were macerated in liquid nitrogen. Frozen powder was immediately transferred into an Eppendorf tube containing 1 mL of lysis buffer (100 mm Tris, pH 8 containing 4% w/v SDS). Lysates were subjected to sonication at 80°C for 40 min, then subjected to further heating at 95°C for 30 min. Cellular debris was removed by centrifugation at 16,100 relative centrifugal force (RCF) RCF for 5 min. The conditions for labeling the lysate by CuAAC for in-gel fluorescence were based on a previously published protocol (Hong et al., 2009). Protein concentrations in the cleared lysate were measured via bicinchoninic acid assay. Forty micrograms of protein lysate was added to PBS to a final total volume of 221 μL. In a separate tube, the alkynyl dye, copper (II) sulfate, and tris(3-hydroxypropyltriazolylmethyl)amine were premixed and allowed to react for 3 min in the dark. Then, aminoguanidine HCl and sodium ascorbate were added to the lysate-PBS mixture. Final concentrations were as follows: alkynyl dye (2.5 μm), CuSO₄ (1 mm), tris(3-hydroxypropyltriazolylmethyl)amine (5 mm), aminoguanidine HCl (5 mm), and sodium ascorbate (5 mm). All components were gently mixed (one inversion) and allowed to react for 15 min in the dark at room temperature without shaking. Proteins were then extracted by methanol/chloroform/water precipitation. Pellets were washed extensively (at least three times). The pellet was then dissolved in 2× SDS sample loading buffer and sonicated for 30 min at 80°C. Samples were heated to 95°C for 5 min and electrophoresed in precast 4% to 12% Bis-Tris polyacrylamide gels. The gel was washed with a fixing solution (50% water, 40% ethanol, 10% acetic acid) in the dark for 10 min and rinsed twice with water (2× 10 min) prior to being subjected to fluorescence imaging with an excitation laser at 532 nm and an emission band pass filter at 580 nm (Typhoon GE Healthcare). After fluorescence imagining, the gel was stained with Colloidal Blue (InstantBlue, Expedeon) for 1 h and imaged to ensure equal protein loading among all lanes. To obtain relative fluorescence values, signal intensity was measured for the entire lane in both the TAMRA (fluorescence) channel and the colloidal blue channel. Next, the ratio of fluorescence intensity to colloidal blue intensity was calculated and normalized to the control lane. Calculating the relative fluorescence values as ratios in this manner allows for comparison between lanes even if lane loading varies slightly.