Donkey anti mouse anti rabbit igg hrp

Transfected cells (approximately 3 × 10⁶ cells) were plated in a 10 cm or 6-well dish and expression was analyzed 24–48 hr after transfection, in parallel to functional assays. Cells were washed with PBS and lysed in RIPA buffer (Amresco N653-100) complemented with a protease inhibitor cocktail (Calbiochem# 539137). After sonication (40%, cycle 2× for 20 s), total protein concentration of the cell lysates was determined by BCA assay (Pierce) and equal amounts of protein were separated by SDS-PAGE using NuPAGE 4–12% Bis-Tris or Tris-Glycine gels. Proteins were electrophoretically transferred to PVDF membranes (Bio-Rad #1704156) or Amersham Protran nitrocellulose membranes (Millipore, #GE10600010) and probed with a monoclonal antibody directed against V5 (clone V5-10, Sigma V8012) or FLAG (clone M2, Sigma F1365), and beta tubulin (Cell Signaling, #2128 (9F3)) or actin (Seven Hills Bioreagents, clone C4, Cincinnati, OH) as loading controls. Blots were then probed with donkey anti-mouse/anti-rabbit IgG HRP (Jackson ImmunoResearch 715-035-150/711-035-152) and analyzed for chemiluminescence on a Bio-Rad reader. Quantification of the bands was performed with Image Lab 6.