Broiler Breeder and Turkey Zona Pellucida Data Blot Links
Description
Link to blots for Westerns for ZPB1, ZPB2 and ZPC
Files
Steps to reproduce
For each sample, 50 µg of IPVL protein lysate was mixed 1:1 with standard sample buffer containing 8 M urea, 2 M thiourea, 3% (wt/vol) SDS, 75 mM DL-dithiothreitol, and 25 mM TrisHCl at pH 6.8, heated at 95°C for 5 m, cooled and loaded into the gel. Fifty micrograms of protein were loaded into each lane of Mini-PROTEAN TGX Stain-Free 10% precast gels (Bio-Rad) alongside Precision Plus Protein All Blue Prestained Protein Standards (Bio-Rad). Gels were subjected to SDS-PAGE in 1X Tris-glycine-SDS (25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS, pH 8.3) running buffer under 70 V for 10 m followed by 120 V for 60 min in a Mini-PROTEAN Tetra Cell system (Bio-Rad). After SDS-PAGE, gels were UV-activated using a ChemiDoc MP Imaging System (Bio-Rad). Proteins were then transferred from the activated gels to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad) in Towbin transfer buffer (25 mM Tris base, 192 mM glycine, and 20% (v/v) methanol) using a wet-blot transfer system (Bio-Rad) at 100V for 1 h. The polyvinylidene difluoride membranes were washed momentarily in Tris-buffered saline (0.02 M Tris base and 0.15 M NaCl, pH 7.4) containing 0.1% Tween 20 (TBST) followed by blocking in 5% BSA (Sigma-Aldrich, St. Louis, MO) in TBST for 30 m. The membranes were then imaged using the ChemiDoc MP Imaging System (BioRad) for total protein normalization to determine relative protein abundance.