Western blot was performed using Midkine Recombinant Rabbit Monoclonal Antibody (JF096-5) (Product # MA5-32538) and an 18 kDa band corresponding to Midkine was observed in conditioned medium of PANC-1 and SH-SY5Y cells but not in conditioned medium of HL-60 and RT4. 7 µl of conditioned media of PANC-1 (Lane 1), SH-SY5Y (Lane 2), HL-60 (Lane 3) and RT-4 (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).