Western blot was performed using Myc Tag Polyclonal Antibody (Product # A-21281) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 60 µg (Lane 1), empty vector control, 60 µg (Lane 2), Myc-p65-V5, 60 µg (Lane 3), Myc-p65-V5, 40 µg (Lane 4), Myc-p65-V5, 20 µg (Lane 5), Myc-p65-V5, 10 µg (Lane 6), FLAG-p65-HA, 40 µg (Lane 7) and 25 ng of Positope (Lane 8) 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). A ~65 kDa band corresponding to Myc-p65-V5 was observed in HEK293E transfected lysates on probing with the primary antibody (0.3 µg/mL) and detected by chemiluminescence with Goat anti-Chicken IgY (H+L) Secondary Antibody, HRP (Product # A16054) . Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Positope (Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags but lacks HA tag, which has been used as a positive control for c-Myc tag detection. No cross-reactivity was seen with FLAG-HA-tagged p65 expressing lysate.
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