Turkey Zona Pellucida Data Blot Links
Link to blots for Westerns for ZPB1, ZPB2 and ZPC
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.