Western blot was performed using Anti-HA Tag Monoclonal Antibody (16B12), Alexa Fluor 594 (Product # A-21288) 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), p65-HA, 40 µg (Lane 3), p65-HA, 20 µg (Lane 4), p65-HA, 10 µg (Lane 5), p65-HA, 5 µg (Lane 6), HA-H3, 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). 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 HEK293E transfected lysates on probing with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 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.
Immunofluorescent analysis of HA Tag was performed using 70% confluent HA-H3 transfected HEK-293E cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with HA Tag Monoclonal Antibody (16B12), Alexa Fluor 594 (Product # A-21288) at 5 µg/mL and Histone H3 Rabbit Polyclonal Antibody (Product # 711055) at 0.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) respectively a dilution of 1:2,000 for 45 minutes at room temperature. Panel a (Nuclei: Red) represents HA Tag Monoclonal Antibody. Panel b (Nuclei: Green) represents Histone H3. Panel c (Nuclei: Blue) represents ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel d represents the merged image showing the co-localization of nuclear signals in transfected cells. Panel e represents untransfected HEK-293E cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Supplier Page from Thermo Fisher Scientific for HA Tag Antibody Alexa Fluor 594