Pierce ecl western blotting substrate detection reagent

Transformed colonies were transferred onto a replica plate and each colony was grown in 600 µL of YPCGal media in a 96-well microplate at 28 °C for 72–96 h at 250 rpm. Cleared supernatants (200 µL) were loaded into a dot blot apparatus connected to a vacuum pump. Pure Pinto-αAI [2 (link)] in sterile YPCGal (1 ng/µL) was used as positive control. Cleared YPCGal inoculated with untransformed cells (200 µL) and sterile YPCGal (200 µL) were used as negative controls. The nitrocellulose membrane was probed with a rabbit anti-αAI polyclonal antibody (1:10,000 dilution), and then probed with HRP-conjugated anti-rabbit secondary antibody (1:1000 dilution, Promega Corp. Madison, WI, USA). Protein antibody complexes were made visible using Pierce ECL Western Blotting Substrate detection reagent (Thermo-Fisher Scientific, Inc., Waltham, MA, USA) and autoradiography film (Kodak Biomax X-ray film). The relative amount of protein in dots was estimated by densitometry using ImageJ Software [38 ].

Western blot samples were prepared by lysing the sciatic nerve tissue in buffer containing [EGTA, pH 8, 150 mM NaCl, 0.5% sodium deoxycholate, 2% (SDS)] and protease inhibitor. Samples were sonicated and centrifuged at 10 000 rpm for 15 min at 4 °C. Supernatants were collected, and the protein concentration was measured using a Pierce BCA protein Assay kit (Thermo Scientific), adjusted to uniform protein content supplemented with an equal volume of Dithiothreitol (DTT) sample buffer, and separated on a 4%–20% SDS-polyacrylamide gel (Mini-Protean TGX, BIO-RAD). Western blotting was performed using primary polyclonal anti-Collagen IV antibody (abcam) and polyclonal anti-Collagen I antibody (abcam) both diluted 1:500 and 1:2000, respectively. Primary antibody against alpha-actin (abcam) was used as a loading control. Secondary peroxidase-conjugated [horseradish peroxidase (HRP)] antibodies (abcam) were diluted 1:2000. Protein detection was performed using Pierce ECL Western blotting substrate detection reagent (32109, Thermo Scientific), and images were acquired using an ImageQuant LAS 4000 luminescence image analyzer (General Electric).