Nupage novex 10 bis tris gel

iNOS protein levels were measured by western blotting. The protein extracts were loaded on a NuPAGE Novex 10 % Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. The membranes were blocked with 5 % bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) for 1 h and then incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody and detected using chemiluminescent HRP substrate (SurModics, Eden Prairie, MN, USA).

The extraction and quantification of the protein from tissues (PEA specimens and pulmonary arteries) [12 ] have been described previously. Protein samples (10 μg) were separated on NuPAGE® Novex 10% Bis-Tris Gel (Invitrogen, Tokyo, Japan) and transferred to nitrocellulose membranes (Invitrogen, Tokyo, Japan). Membranes were blocked with 5% non-fat dried milk in PBS containing 0.5% Tween20 for 1 h at room temperature, and were then incubated with primary antibodies overnight at 4 °C. The membranes were incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature. Chemiluminescence was detected using a LAS-4000 (Fuji Film, Tokyo, Japan). The blots were scanned and a densitometry analysis was conducted using the Image J software program.

For MALDI-MS/MS protein identification, purified RcGTA samples were run on a TEO-Tricine 4–12% SDS minigel (Expedeon) at 150 V for 45 min. Gels were stained with InstantBlue protein stain (Expedeon) for 1 h before destaining with ultrapure water for 1 h. Protein bands of interest were excised. For shotgun LC-MS/MS, samples were run on a 7-cm NuPAGE Novex 10% bis-Tris gel (Life Technologies) at 200 V for 6 min. Gels were stained with SafeBLUE protein stain (NBS biologicals) for 1 h before destaining with ultrapure water for 1 h.
In-gel tryptic digestion was performed after reduction with dithioerythritol and S-carbamidomethylation with iodoacetamide. Gel pieces were washed two times with 50% (vol/vol) aqueous acetonitrile containing 25 mM ammonium bicarbonate and then once with acetonitrile and then dried in a vacuum concentrator for 20 min. Sequencing-grade modified porcine trypsin (Promega) was dissolved in the 50 mM acetic acid supplied by the manufacturer and then diluted 5-fold by adding 25 mM ammonium bicarbonate to give a final trypsin concentration of 0.02 μg/μl. Gel pieces were rehydrated by adding 10 μl of trypsin solution, and after 5 min, enough 25 mM ammonium bicarbonate solution was added to cover the gel pieces. Digests were incubated overnight at 37°C.

Whole cell lysates were separated on a NuPAGE^® Novex^® 10% Bis-Tris Gel (Life Technologies Corporation) and transferred to PVDF membrane. The membrane was blocked with Tris-buffered saline (pH 7.5) containing 0.2% Tween 20 and 5% nonfat dry milk. Primary antibodies used were as follows: rabbit anti-DCK (GeneTex, Inc. CA) and anti-Gapdh (1:10,000, Cell Signaling Technology, Danvers, MA). Goat anti-Rabbit HRP conjugated secondary antibodies were utilized at 1:5000 dilution (Santa Cruz Biotechnologies, Dallas, TX). Membranes were developed with Western Bright Ecl kit (BioExpress, Kaysville, UT).

Triplicate biological samples were solubilized in NuPAGE LDS sample buffer (Life Technologies), heated at 70 °C for 10min and ran on a 7 cm NuPAGE Novex 10% Bis-Tris gel (Life Technologies) at 200 V for 6min. Gels were stained with SafeBLUE protein stain (NBS biologicals) for 1 h before destaining with ultrapure water for 1 h. In-gel tryptic digestion was performed after reduction with dithioerythritol and S-carbamidomethylation with iodoacetamide. Gel pieces were washed two times with aqueous 50% (v:v) acetonitrile containing 25 mm ammonium bicarbonate, then once with acetonitrile and concentrated in a vacuum for 20min. Sequence-grade, modified porcine trypsin (Promega) was dissolved in 50 mm acetic acid and diluted with 25 mm ammonium bicarbonate to give a final trypsin concentration of 0.02g/L. Gel pieces were rehydrated with 25 L of trypsin solution, incubated for 10 min then 25 mm ammonium bicarbonate solution was added to cover the gel pieces. Digests were incubated overnight at 37 °C. Peptides were extracted by washing three times with aqueous 50% (v:v) acetonitrile containing 0.1% (v:v) trifluoroacetic acid, before concentrating in a vacuum and reconstituting in aqueous 0.1% (v:v) trifluoroacetic acid. A common sample pool was created by taking equal aliquots of all samples.