2d electrophoresis buffer  (Bio-Rad)

Journal: PLoS ONE

Article Title: Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

doi: 10.1371/journal.pone.0080693

Figure Lengend Snippet: Approximately 40 mg of total protein from (A) eggs, (B) isolated cortical fragments, (C) 16-cell stage embryos, (D) 16-cell stage micromeres, were collected from S . purpuratus and then subjected to 2D gel electrophoresis, separating the proteins first by charge (on a pH 4 to pH 7 IPG strip) then by size, and then to Western blot analysis using anti-Dsh antibodies. Dsh (red) isoforms in isolated cortex samples and micromere samples are enriched near the acidic (left) side of the gel, while whole embryo samples show the presence of Dsh protein species that are not detected in the isolated cortex and 16-cell stage micromeres. Tubulin (green) serves as an internal control.

Article Snippet: For 2D SDS-PAGE 40 μg of total protein of each sample was solubilized in 125 μl of 2D electrophoresis buffer (ReadyPrep 2D Rehydration/Sample Buffer 1; 7 M urea, 2 M thiourea, 1% (w/v) ASB-14, 40 mM Tris, 0.001% Bromophenol Blue, BIO-RAD) with the addition of 25 mM DTT, 4% (w/v) CHAPS, and 0.2% Bio-Lyte pH 3-10/4-7 (2:1)(BIO-RAD).

Techniques: Isolation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Stripping Membranes, Western Blot, Control