Tris 2 carboxyethyl phosphine hydrochloride

HEK293 cells transiently expressing GFP-tagged claudin were labeled with 25 µM 17-octadecynoic acid (Sigma-Aldrich, O8382) in a normal culture medium for 24 h. The cells were washed with PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors). Lysates were incubated with anti-GFP antibody overnight at 4 °C. Protein G Sepharose (GE Healthcare) was added to lysate and incubated for 3 h. Immunoprecipitated GFP-tagged proteins were eluted from the beads with 1% SDS, 50 mM HEPES pH −7.4 and adjusted to 1 mM CuSO4 (Wako, 030-04442), 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich Co., C4706), 100 µM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (Tokyo Chemical Industry, T2993), and 100 µM TAMRA-Azide-Biotin (Click Chemistry Tools, #1048). The reaction mixture was gently shaken at RT in darkness for 1 h. The click reactions were stopped by adding 10 volumes of ice-cold methanol, and proteins were precipitated overnight at −80 °C. The precipitated samples were spun down at 15,000 rpm at 4 °C for 30 min, rinsed in ice-cold methanol, and eluted in SDS sample buffer for analysis by SDS-PAGE. Gels were scanned with an Amersham Typhoon scanner (GE Healthcare).

Proteins were precipitated in 23% trichloroacetic acid (product number T-0699, Sigma-Aldrich) at 4°C overnight. After 30-min centrifugation at 18,000g, protein pellets were washed two times with 500 μl of ice-cold acetone. Air-dried pellets were dissolved in 8 M urea/100 mM tris (pH 8.5). Proteins were reduced with 1 M tris(2-carboxyethyl)phosphine hydrochloride (product number C4706, Sigma-Aldrich) and alkylated with 500 mM 2-chloroacetamide (product number 22790-250G-F, Sigma-Aldrich). Proteins were digested for 18 hours at 37°C in 2 M urea, 100 mM tris (pH 8.5), and 1 mM CaCl₂ with 2 μg of trypsin (product number V5111, Promega). Digestion was stopped with formic acid, 5% final concentration. Debris was removed by centrifugation at 18,000g for 30 min.