Immunofluorescent analysis of 6x-His Tag was performed using 70% confluent HEK-293 cells transfected with V5-H3-His construct. 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 6x-His Tag Polyclonal Antibody (Product # PA1-983B) at 5 µg/mL and Histone H3 Monoclonal Antibody (865R2) (Product # AHO1432) at 1:200 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 (H+L)/IgM (L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjµgate (Product # A28175) respectively at a dilution of 1:2000 for 45 minutes at room temperature. Panel a (Nuclei: Red) represents 6x-His Tag Polyclonal Antibody. Panel b (Nuclei: Green) represents Histone H3 Monoclonal Antibody (865R2). Panel c (Nuclei: Blue) represents nuclei stained wirh 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 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Western Blot was performed using Anti-6x-His Tag Polyclonal Antibody (Product # PA1-983B) 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), V5-H3-His, 60 µg (Lane 3), V5-H3-His, 40 µg (Lane 4), V5-H3-His, 20 µg (Lane 5), Myc-p65-V5, 40 µg (Lane 6) and 25 ng Positope (Lane 7) 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 17 kDa band of V5-H3-His were observed in transfected lysates on probing with primary antibody (0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) 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, which has been used as a positive control for 6x-His Tag detection. No cross-reactivity was seen with Myc-p65-V5 expressing lysate.