![]() ![]() ![]() No cross-reactivity was seen with p65 expressing lysate. Positope (Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags along with V5 tag, which has been used as a positive control for V5 tag detection. A ~65 kDa band corresponding to Myc-p65-V5 were observed in HEK293E transfected lysates on probing with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) 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 V5 Tag Monoclonal Antibody (Product # R960-25) 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), V5-H3-His, 50 µg (Lane 3), Myc-p65-V5, 50 µg (Lane 4), Myc-p65-V5, 25 µg (Lane 5), Myc-p65-V5, 12.5 µg (Lane 6), Myc-p65-V5, 6.26 µg (Lane 7), p65-HA, 50 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 FLAG-tagged p65 expressing lysate. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A ~19 kDa band corresponding to V5-H3-His and ~65 kDa band corresponding to Myc-p65-V5 were observed in HEK293E transfected lysates on probing with the primary antibody (1 µg/mL) 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). Western blot was performed using V5 Tag Monoclonal Antibody (Product # R960-25) 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), V5-H3-His, 50 µg (Lane 3), Myc-p65-V5, 50 µg (Lane 4), Myc-p65-V5, 25 µg (Lane 5), Myc-p65-V5, 12.5 µg (Lane 6), Myc-p65-V5, 6.26 µg (Lane 7), FLAG-P65-HA, 50 µg (Lane 8) and 25 ng of Positope (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). The images were captured at 60X magnification. Panel f represents control cells with no primary antibody to assess background. Panel e represents untransfected HEK-293 cells. Panel d represents the merged image showing the co-localization of nuclear signals in transfected cells. Panel c (Nuclei: Blue) represents ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel b (Nuclei: Green) represents Histone H3. The cells were labeled with V5 Tag Monoclonal Antibody (Product # R960-25) at 1:100 dilution and Histone H3 Recombinant Polyclonal Antibody (Product # 711055) at 0.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Product # A32728) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) respectively at a dilution of 1:2000 for 45 minutes at room temperature. 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. Immunofluorescent analysis of V5 Tag was performed using 70% confluent V5-H3-6XHis transfected HEK-293 cells. ![]()
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