The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 48 kDa (predicted molecular weight: 44 kDa).
Cysteine protease required for autophagy, which cleaves the C-terminal part of either MAP1LC3, GABARAPL2 or GABARAP, allowing the liberation of form I. A subpopulation of form I is subsequently converted to a smaller form (form II). Form II, with a revealed C-terminal glycine, is considered to be the phosphatidylethanolamine (PE)-conjugated form, and has the capacity for the binding to autophagosomes.
Mainly expressed in the skeletal muscle, followed by brain, heart, liver and pancreas.
Lane 1: Wild type HAP1 whole cell lysate (20 µg) Lane 2: ATG4B knockout HAP1 whole cell lysate (20 µg) Lane 3: HeLa whole cell lysate (20 µg)
Lanes 1 - 3: Merged signal (red and green). Green - ab72878 observed at 44 kDa. Red - loading control, ab18058, observed at 130 kDa.
ab72878 was shown to recognize ATG4B when ATG4B knockout samples were used, along with additional cross-reactive bands. Wild-type and ATG4B knockout samples were subjected to SDS-PAGE. ab72878 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
Western blot - Anti-ATG4B antibody (ab72878)
All lanes : Anti-ATG4B antibody (ab72878) at 1 µg/ml
Lane 1 : Human heart tissue lysate - total protein (ab29431) Lane 2 : Human liver tissue lysate - total protein (ab29889)
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution