Positope ( Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags but lacks DYKDDDDK tag, which has been used as a negative control for DYKDDDDK tag detection. A ~65 kDa band corresponding to DYKDDDDK-p65-HA were observed in HEK293E transfected lysates on probing with the primary antibody (1 µg/mL) and detected by fluorescence using the iBright FL 1500 ( Product # A44241). Resolved proteins were then transferred onto a nitrocellulose membrane ( Product # IB23001) by iBlot® 2 Dry Blotting System ( Product # IB21001). Western blot was performed using Anti-DYKDDDDK Tag Monoclonal Antibody (L5), Alexa Fluor 488 ( Product # MA1-142-A488) 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-DYKDDDDK, 40 µg (Lane 3), DYKDDDDK-p65-HA, 40 µg (Lane 4), DYKDDDDK-p65-HA, 20 µg (Lane 5), DYKDDDDK-p65-HA, 10 µg (Lane 6), DYKDDDDK-p65-HA, 5 µg (Lane 7), Myc-p65-V5, 40 µg (Lane 8) and 25 ng of Positope (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel ( Product # NP0322BOX). No cross-reactivity was seen with Myc or V5-tagged p65 expressing lysate. Positope ( Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags but lacks FLAG tag, which has been used as a negative control for FLAG tag detection. ![]() Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit ( Product # WP20005). A ~65 kDa band corresponding to FLAG-p65-HA were observed in HEK293E transfected lysates on probing with the primary antibody (1 µg/mL) and detected by fluorescence using the iBright FL 1500 ( Product # A44241). ![]() Western blot was performed using Anti-DYKDDDDK Tag Monoclonal Antibody (L5), Alexa Fluor 488 ( Product # MA1-142-A488) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 50 µg (Lane 1), empty vector control, 50 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 of Positope (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel ( Product # NP0322BOX).
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