Western blot was performed using Anti-HA Tag Polyclonal Antibody (Product # PA1-985) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 40 µg (Lane 1), empty vector control, 40 µg (Lane 2), HA-H3, 40 µg (Lane 3), HA-H3, 20 µg (Lane 4), HA-H3, 10 µg (Lane 5), HA-H3, 5 µg (Lane 6), p65-HA, 40 µg (Lane 7), Myc-p65-V5, 40 µg (Lane 8 and 25 ng Positope (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). A ~19 kDa band corresponding to HA-H3 and ~67 kDa band corresponding to p65-HA were observed in transfected lysates on probing with primary antibody (1:2000 dilution) and and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1500 (Product # A44241). Positope (Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags but lacks HA tag, which has been used as a negative control for HA tag detection. No cross-reactivity was seen with Myc or V5-tagged p65 expressing lysate.
Western blot was performed using Anti-HA Tag Polyclonal Antibody (Product # PA1-985) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 40 µg (Lane 1), empty vector control, 40 µg (Lane 2), HA-H3-FLAG, 40 µg (Lane 3), HA-H3-FLAG, 20 µg (Lane 4), HA-H3-FLAG, 10 µg (Lane 5), HA-H3-FLAG, 5 µg (Lane 6), FLAG-p65-HA, 40 µg (Lane 7), Myc-p65-V5, 40 µg (Lane 8 and 25 ng Positope (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). A ~19 kDa band corresponding to HA-H3-FLAG and ~67 kDa band corresponding to FLAG-p65-HA were observed in transfected lysates on probing with primary antibody (1:2000 dilution) and and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1500 (Product # A44241). 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 negative control for HA tag detection. No cross-reactivity was seen with Myc or V5-tagged p65 expressing lysate.
Supplier Page from Thermo Fisher Scientific for HA Tag Antibody