Left: Flow cytometry analysis of human SHSY-5Y cells of MAP1LC3A with M-1725-100. Fixing and Permeabilization of cells: Absolute methanol and 0.1% Tween-20 in PBS, Primary antibody: Mouse Monoclonal antibody to MAP1LC3A (cat # M-1725-100, 2 μg per ~10^6 cells) for 15 minutes at room temperature, Secondary antibody: Goat anti-mouse PE labelled secondary antibody (1:1000 fold dilution) with incubation for 15 minutes in dark at room temperature. Non-specific Control IgG, clone X63 (cat # M-1249-200) was used under same conditions as negative control (black dashed). Flow cytometry data and results were generated using Orflo Moxiflow instrument and protocols. The data demonstrates specific staining of MAP1LC3A expressed in human neuronal cell line SHSY-5Y using cat # M-1725-100.
Right: Detection of MAP1LC3A in human SHSY-5Y cell lysates (20 µg/lane) by Western Blotting. SDS-PAGE: 4-20% Bis-Tris denatured, reduced; Transfer: Tris-Glycine buffer; Membrane: nitrocellulose (0.2 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4°C (10 µg/mL); Secondary antibody: anti-mouse-HRP (1/10000) 2 hours at RT; Detection: Enhanced Chemiluminescence Substrate with LiCor C-DiGit Blot Scanner. The broad band seen on WB indicates binding of Cat # M-1725-100 to a broad protein band with a Molecular Weight of ~14 kDa. Predicted molecular weight of MAP1LC3A is ~14.5 kDa.