Goat anti mouse igg secondary antibody hrp

Protein lysates were obtained from 300k cells per sample using RIPA buffer supplemented with protease and phosphatase inhibitors. To prepare Western blot samples, protein solutions were mixed with Laemmli buffer (2X, contain 5 %v/v β-mercapto ethanol, bio-rad) in 1:1 ratio and heated at 97°C for 5 min. Samples were loaded in a Nupage™ 4–12% bis-Tris protein gel (Thermoscientific). Gels were run in MOPS-SDS buffer (20X, thermo fisher) at 60V for 15 min the 165V for 1 h. Later proteins were transferred onto a PVDF membrane (Thermo Scientific™) in tris/glycine (10X, Bio-Rad) buffer containing 20 %v/v methanol. IBSP detection was done using a human IBSP polycolonal antibody (Rabbit, Thermo Fisher Scientific) as the primary antibody and then a goat anti-rabbit IgG (HRP linked, cell signaling). For the housekeeping gene, GAPDH was stained using a GAPDH loading control antibody (mouse, Fisher Scientific) and then a goat anti-mouse IgG secondary antibody (HRP, Novus biological). Protein bands were developed using the Clarity™ western ECL substrate (Bio-Rad). Protein bands were visualized using MYECL gel imager (Thermo Scientific). Quantification of protein levels were done in ImageJ.