Western Blot was performed using Anti-GFP Monoclonal Antibody (GF28R), DyLight 680 (Product # MA5-15256-D680) 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). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). 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 (1:1000 dilution) and detected by fluorescence using the iBright FL 1500 (Product # A44241). Positope (Product # R90050) is a 53 kDa recombinant protein consisting multiple epitope tags, which has been used as a positive control for GFP detection. This product also detects Yellow Fluorescent Protein (YFP), a variant of GFP as observed in Lane 7. No cross-reactivity was seen with mCherry (RFP family) expressing lysate.
Immunofluorescent analysis of GFP Tag was performed using H3-GFP Tag transfected 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 1 hour at room temperature. The cells were labeled with GFP Monoclonal Antibody (GF28R), DyLight 680 (Product # MA5-15256-D680) 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-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (Product # A32732) at a dilution of 1:2000 for 45 minutes at room temperature. Panel a (Nuclei: Green) represents GFP Monoclonal Antibody (GF28R), DyLight 680, a green pseudo-colour has been given after image capture. Panel b (Nuclei: Red) represents Histone H3 Recombinant Polyclonal Antibody. Panel c (Nuclei: Blue) represents ProLongTM 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.
Supplier Page from Thermo Fisher Scientific for GFP Antibody DyLight 680