Hemoglobin β γ δ ε sc 390668

Approximately 10⁶ cells were harvested, by centrifugation at 300×g for 5 min, and processed for total protein isolation and western blot analysis. Pelleted cells were washed twice with PBS and lysed in buffer A (10 mM Tris-Cl pH 8.0, 150 mM NaCl, 2% SDS). The lysate appeared highly viscous due to DNA release and, therefore, it was sonicated for 30 s. The samples were then processed for protein quantitation using the BCA assay kit. One volume of protein sample was, consequently, mixed with equal volume of laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris HCl, pH 6.8) and boiled for 5 min at 95 °C. Twenty µg of total protein material was then loaded on a denaturing 12% SDS-PAGE gel and run until separation. Subsequently, proteins were transferred to a PVDF membrane and blotted with primary antibodies overnight at 4 °C in a shaker and with secondary antibody for 1 h at room temperature. The antibodies used were from Santa Cruz Biotechnology (Finnell Street Dallas, Texas 75220, USA): Hemoglobin β/γ/δ/ε (sc-390668), β-actin (sc-47778), α- tubulin (sc-51503) and m-IgGκBP-HRP (sc-516102).

Approximately 10⁶ cells of each cell culture were harvested, by centrifugation at 300× g for 5 min, and processed for total protein isolation and Western blot analysis. Pelleted cells were washed with PBS and lysed in lysis buffer (10 mM Tris-Cl pH 8.0, 150 mM NaCl, 2% SDS). The lysate was highly viscous due to DNA release and, therefore, it was sonicated for 30 s. The samples were then processed for protein quantitation using the BCA assay kit (ThermoFisher Scientific, Waltham, MA, USA). After protein quantification, one volume of protein sample was mixed with equal volume of Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris HCl, pH 6.8) and heated for 5 min at 95 °C. Twenty micrograms of total protein material was loaded on a denaturing 12% SDS-PAGE gel and run until separation. Proteins were, then, transferred to a PVDF membrane and blotted with primary antibodies overnight at 4 °C and with secondary antibody for 1 h at room temperature. The antibodies used were from Santa Cruz Biotechnology (Dallas, TX, USA): Hemoglobin β/γ/δ/ε (sc-390668, diluted 1:200), β-actin (sc-47778, diluted 1:500), Rps6 (sc-74459, diluted 1:500) and m-IgGκBP-HRP (sc-516102, diluted 1:5000).