Anti-EGFP antibody [F56-6A1.2.3]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EGFP antibody [F56-6A1.2.3] (AB184601)

Immunofluorescent analysis of GFP Tag was performed using H3-GFP construct transfected in HEK-293E 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 ab184601 at 1/100 dilution and Histone H3 Rabbit Polyclonal Antibody at 0.5 μg/mL in 0.1% BSA, incubated at 4°C overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor^® Plus 555 and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor^® Plus 647 respectively at a dilution of 1/2000 for 45 minutes at room temperature.

Image a : Anti-EGFP antibody [F56-6A1.2.3], ab184601 (green)
Image b : Histone H3 (red)
Image c : Nuclei stained with DAPI (blue)
Image d : Merged image showing the co-localization of nuclear signals in transfected cells
Image e : Untransfected HEK cells
Image f : Control cells with no primary antibody to assess background

The images were captured at 60X magnification.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EGFP antibody [F56-6A1.2.3] (AB184601)

Immunofluorescent analysis of U-2 OS (Human bone osteosarcoma epithelial cell line) cells transfected with an EGFR-EGFP fusion protein (formalin-fixed, 0.1% Triton X-100 permeabilized) using either natural fluorescence (green) or an anti-EGFP antibody (red). Cells were then labeled with ab184601 at 1/20 dilution followed with DyLight 550 goat anti-mouse IgG secondary antibody at 1/200 dilution. Nuclei (blue) were stained with Hoechst 33342 dye. 20X magnification.

• WB

Supplier Data

Western blot - Anti-EGFP antibody [F56-6A1.2.3] (AB184601)

All lanes:

Western blot - Anti-EGFP antibody [F56-6A1.2.3] (ab184601) at 1/1000 dilution

Lane 1:

Recombinant GFP at 1 µg

Lane 2:

Recombinant GFP at 0.5 µg

Lane 3:

Recombinant GFP at 0.25 µg

Lane 4:

Recombinant GFP at 0.125 µg

Lane 5:

Recombinant GFP at 0.0625 µg

Secondary

All lanes:

goat anti-mouse IgG-HRP at 1/15000 dilution

Predicted band size: 27 kDa

true

• WB

Supplier Data

Western blot - Anti-EGFP antibody [F56-6A1.2.3] (AB184601)

Western Blot was performed using ab184601 by loading whole cell extracts of untransfected and transiently transfected HEK-293E. Lysates were electrophoresed using a 4-12% Bis-Tris Protein Gel. Resolved proteins were then transferred onto a nitrocellulose membrane using a dry blotting system. A ~45 kDa band of H3-GFP and a ~92 kDa band of p65-GFP were observed in transfected lysates. A 53 kDa recombinant protein consisting multiple epitope tags was 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.

All lanes:

Western blot - Anti-EGFP antibody [F56-6A1.2.3] (ab184601) at 1/1000 dilution

Lane 1:

Untransfected HEK-293E (human epithelial cell line from embryonic kidney) whole cell lysate at 40 µg

Lane 2:

Empty vector control at 40 µg

Lane 3:

H3-GFP at 40 µg

Lane 4:

H3-GFP at 20 µg

Lane 5:

H3-GFP at 10 µg

Lane 6:

p65-GFP at 40 µg

Lane 7:

p65-YFP at 40 µg

Lane 8:

H3-mCherry at 40 µg

Lane 9:

53 kDa recombinant protein consisting multiple epitope tags at 0.025 µg

Secondary

All lanes:

Goat anti-Mouse IgG (H+L) (HRP) at 1/4000 dilution

Predicted band size: 27 kDa

true