HA Tag Antibodies

Western blot analysis was performed using 90 µg (Lane 1), 60 µg (Lane 2), 30 µg (Lane 3) of whole cell lysates of HEK-293T cells transfected with an HA tagged Histone H3 vector, or left untransfected (Lane 4). The blots were probed with HA Tag Monoclonal Antibody (2-2.2.14) (Cat. No. 26183, 0.2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A28177, 0.4 µg/mL, 1:2,500 dilution). A ~17 kDa band corresponding to HA tagged Histone H3 was observed in the transfected samples. Known quantity of protein samples were electrophoresed using NuPAGE 10 % Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well (Cat. No. NP0302BOX), XCell SureLock Electrophoresis System (Cat. No. EI0002), and Novex Sharp Pre-Stained Protein Standard (Cat. No. LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot 2 Gel Transfer Device (Cat. No. IB21001). The membrane was probed with the relevant primary and secondary antibody using iBind Flex Western Starter Kit (Cat. No. SLF2000S). Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Cat. No. 32106).

Immunofluorescence analysis of HA Tag was performed using HEK-293 cells transfected with a HA tagged Histone H3 vector. After 48 hours, 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 (2-2.2.14) (Cat. No. 26183) at a concentration of 2 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 488 (Cat. No. A32723) at a dilution of 1:2,000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d represents the merged image showing nucleus localization. Panel e shows untransfected control cells and panel f represents cells with no primary antibody to assess background. The images were captured at 60X magnification.