Rabbit anti human gapdh monoclonal antibody

Cell lysis buffer and a protease inhibitor cocktail were added to the cell pellet, and a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL USA) was used to determine protein concentration.
For SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the sample volume to be loaded was calculated based on the protein concentration (30 µg protein). Proteins were separated via SDS-PAGE, transferred to a polyvinylidene fluoride membrane, blocked in skim milk at room temperature for 1 h, and incubated with diluted mouse anti-human Wnt5a monoclonal antibody (1:5,000), rabbit anti-human H4K20me1 monoclonal antibody (1:10,000) (both from Millipore, Billerica, MA, USA), or rabbit anti-human GAPDH monoclonal antibody (1:5,000; Abcam, Cambridge, MA, USA) overnight at 4̊C. Next, the membrane was washed with Tris-buffered saline with Tween-20 (TBST) (10 min, ×3) and incubated with horseradish peroxidase-labeled goat anti-mouse antibody or goat anti-rabbit secondary antibody for 1 h. An equal volume of reagent A and B of an ECL luminescent kit was mixed and evenly spread on the membrane. Subsequently, the membrane was exposed, developed and fixed to Kodak film in the darkroom; Quantity One software (Bio-Rad Laboratories, Hercules, CA USA) was used for the quantitative analysis, using GAPDH as an internal reference. The experiment was repeated 3 times.