Bicinchoninic acid bca method

3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 4 mM glutamine, 4500 mg/liter glucose, 1 mM sodium pyruvate, and 1500 mg/liter sodium bicarbonate. To maintain cells in a pre-differentiated state, they were not allowed to reach confluence. Differentiation of 3T3-L1 cells into adipocytes was initiated by the addition of 10 μM dexamethasone, 0.5 mM isobutylmethylxanthine, and 10 μg/ml insulin to confluent cells for 2 days. The cells were then cultured in medium containing 10 μg/ml insulin for 2 days after which they were maintained in complete media. The medium was changed every 2 days for 6 to 8 days, at which time differentiation was complete.
3T3-L1 cells were solubilized in 20 mM HEPES (pH 7.4), 1.0 mM EGTA, 1% Triton X-100, and 10% glycerol on ice for 20 min. The extracts were then centrifuged at 4°C for 5 min at 14,000 x g in an Eppendorf microfuge. The supernatant was recovered, and protein concentration was determined using the bicinchoninic acid (BCA) method (Thermo Fisher Scientific, Waltham, MA). Aliquots were taken for SDS-PAGE as described below.

All operations were carried out at 4°C. The seed cotyledons weighing 91 g were homogenized in 100 mL extraction buffer (50 mM NaOAc, pH 5.0, 150 mM NaCl and 10 mM MgCl₂) using polytron tissuemizer (Tekmar Tissumizer MarkII, Cincinnati, OH) at low, medium and high speed for 30 s each. The homogenate was cooling down on ice for 1 min between bursts. The homogenate was centrifuged at 3,000 g for 15 min and the resulting supernatant (S1) was centrifuged at 18,000 g for 30 min at 4°C (Sorvall RC2B, Miami, FL). This supernatant (S2) was ultracentrifuged at 105,000 g for 60 min at 4°C (Sorvall Discovery 100SE, Hitachi Ltd, Tokyo, Japan) and the resulting supernatant (S3, cytosol) and the pellet (P3, microsomal membranes) were collected. The S3 supernatant was dialyzed to remove Pi from the seed extract against imidazole buffer (25 mM imidazole buffer, pH 6.5, 1 mM MgCl₂) with three 500 mL buffer changes. The dialyzed supernatant became cloudy after dialysis, which was removed by centrifugation at 18,000 g for 30 min at 4°C. The final supernatant (S3-supernatant) and pellet (S3-pellet) as well as P3 were used for determining subcellular distribution of PAP activity and S3-supernatant was used for PAP purification. The protein content of the supernatant was determined by Bicinchoninic acid (BCA) method (Thermo Scientific, Rockford, IL).

Cells were lysed with a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany), and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA)] and centrifuged at 20,000 × g for 20 min at 4°C. The supernatants were collected, and the protein concentrations were quantitated with the bicinchoninic acid (BCA) method (Thermo Fisher Scientific, San Jose, CA, USA) using bovine serum albumin as the standard. The lysates were then solubilized with immunoblot sample buffer [46.7 mM Tris-HCl (pH 6.8), 5% glycerol, 1.67% SDS, 1.55% dithiothreitol, and 0.003% bromophenol blue]. Next, the samples were separated by SDS-PAGE, transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany), and blotted with primary and secondary antibodies. Each protein signal was detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Darmstadt, Germany). The signal intensities were captured with the LuminoGraph image analyzer (ATTO, Tokyo, Japan) and the images were processed using Photoshop CS6 software (Adobe Systems Corporation, San Jose, CA, USA).