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As a fusion tag, GFP can be used to localize proteins, to study their movement or to research the dynamics of the subcellular compartments where these proteins are targeted. GFP has been used widely as a reporter protein for gene expression in eukaryotic and prokaryotic organisms, and as a protein tag in cell culture and in multicellular organisms. The jellyfish Aequorea victoria contains green fluorescent protein (GFP Tag) that emits light in the bioluminescence reaction of the animal. No cross-reactivity was seen with mCherry (RFP family) expressing lysate. This product also detects Yellow Fluorescent Protein (YFP), a variant of GFP as observed in Lane 7. Positope ( Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags, which has been used as a positive control for GFP detection. A ~45 kDa band of H3-GFP and a ~92 kDa band of p65-GFP were observed in transfected lysates on probing with primary antibody (1:2000 dilution) and and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ 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 Anti-GFP Polyclonal Antibody ( Product # A-11122) 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), H3-GFP, 40 µg (Lane 3), H3-GFP, 20 µg (Lane 4), H3-GFP, 10 µg (Lane 5), p65-GFP, 40 µg (Lane 6), His-p65-YFP, 40 µg (Lane 7), H3-mCherry, 40 µg (Lane 8) and 25 ng 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 cells. Panel d represents the merged image showing the co-localization of nuclear signals in transfected cells. Panel c (Nuclei: Blue) represents ProLongTM Diamond Antifade Mountant with DAPI ( Product # P36962). Panel b (Nuclei: Red) represents Histone H3. Panel a (Nuclei: Green) represents GFP Polyclonal Antibody, a green pseudo-colour has been given after image capture. The cells were labeled with GFP Polyclonal Antibody ( Product # A-11122) at 1:200 dilution and Histone H3 Monoclonal Antibody (865R2) 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 555 ( Product # A32732) and Goat anti-Mouse IgM (Heavy Chain) Secondary Antibody, Alexa Fluor 647 ( Product # A-21238) 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 GFP Tag was performed using H3-GFP construct transfected in HEK-293E cells.

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