Western blot was performed using Anti-Myc Tag Polyclonal Antibody (Product # PA1-981) 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), His-H3-Myc, 60 µg (Lane 3), His-H3-Myc, 40 µg (Lane 4), His-H3-Myc, 20 µg (Lane 5), His-H3-Myc, 10 µg (Lane 6), Myc-p65-V5, 60 µg (Lane 7,) p65-HA, 60 µg (Lane 8) and 25 ng of Positope (Lane 9) 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 and ~17 kDa band corresponding to His-H3-Myc was observed in HEK293E transfected lysates on probing with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP (Product # A27036). 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 p65-HA expressing lysate.
Immunofluorescence analysis of Myc proto-oncogene protein was performed using 70% confluent log phase HEK-293 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 45 minutes at room temperature. The cells were labeled with Myc Tag Polyclonal Antibody (Product # PA1-981) at 1:100 in 0.1% BSA and Histone H3 antibody (Product # AHO1432) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Product # A32733) and Goat anti-Mouse IgG Alexa Fluor Plus 488 (Product # A21042) respectively at a dilution of 1:2,000 for 45 minutes at room temperature. Panel a (Nuclei: Red) represents Myc tag. Panel b (Nuclei: Green) represents Histone 3. 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 un-transfected HEK-293 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
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