All lanes : Anti-GFP antibody (ab13970) at 1/2000 dilution (Diluent 1x TBS /4 hours at 4°C)

Lane 1 : 3 µg of GFP plasmid overexpressed in mouse cardiomyocytes whole cell lysate with BSA / for 1 hour at room temperature
Lane 2 : 2 µg of GFP plasmid overexpressed in mouse cardiomyocytes whole cell lysate with BSA / for 1 hour at room temperature
Lane 3 : 1 µg of GFP plasmid overexpressed in mouse cardiomyocytes whole cell lysate with BSA / for 1 hour at room temperature

Lysates/proteins at 25 µg per lane.

Blocking peptides at 5 % per lane.

Secondary
All lanes : Goat Anti-Chicken IgY H&L (Alexa Fluor® 594) preadsorbed (ab150176) at 1/5000 dilution

Performed under reducing conditions.

Additional bands at: 25 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 30 seconds

Gel Running Conditions: Reduced Denaturing (15% PAGE)

Detection method: Fluorescent Secondary Antibodies

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ab13970 staining GFP in U-2 OS (Human bone osteosarcoma epithelial cell line) cells by ICC/IF.

Cells were paraformaldehyde fixed, permeabilized with 0.5% triton and blocked with 2% antibody dilution buffer for 2 hours. Cells were incubated with the primary antibody (1/1000) for 1 hour at 25°C. An undiluted Alexa Fluor^® 488 conjugated Goat anti-chicken polyclonal was used as the secondary antibody.

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All lanes : Anti-GFP antibody (ab13970) at 1/1000 dilution

Lane 1 : Whole cell lysate prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.
Lane 2 : Whole cell lysate prepared from HeLa cells.

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : IRDye 800CW conjugated goat anti-chicken polyclonal at 1/15000 dilution

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Transgenic mice expressing GFP selectively in lamina II of the spinal cord.

In the right panels, note the correspondance between the green (rabbit anti-GFP) and red signals (chicken anti-GFP from Abcam) indicating that these two antibody preparations recognized the same gene product. The secondary antibody used with ab13970 was an FITC-labeled goat anti-chicken

ab13970 staining GFP in GFP-transfected NIH/3T3 (Mouse embryo fibroblast cell line) cells.

The cells were fixed with 4% formaldehyde (10 minutes) and then blocked in 1% BSA / 0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab13970 at 1/2000 dilution overnight at +4°C followed by incubation with Goat Anti-Chicken IgY H&L (Alexa Fluor^® 488) preadsorbed (ab150173), for 1 hour, at 1 μg/ml.
Under identical experimental conditions, when compared to the basal level of GFP expression in transfected NIH/3T3 cells, the cells upon which ab13970 was applied gave a stronger signal in the 488 channel, indicating that ab13970 is binding to GFP and therefore eliciting signal amplification.
ab13970 was also applied to non-GFP-transfected NIH/3T3 cells, which produced no positive staining, indicating specificity for GFP.

Nuclear DNA was labeled with 1.43 μM DAPI (blue).

Lane 1 : Rabbit anti-GFP
Lane 2 : Anti-GFP antibody (ab13970)

All lanes : Transgenic mouse spinal cords

Western blot of transgenic mouse spinal cords showing that the rabbit anti-GFP (lane 1) and the chicken anti-GFP (Abcam; lane 2) recognize a band at the same molecular weight.

ab13970 staining GFP + tumor in mouse muscle cells by ICC/IF.

Cells were formaldehyde fixed and blocked with 3% BSA for 1 hour at 24°C prior to incubation with the primary antibody (1/500) for 1 hour at 24°C. An Alexa Fluor^® 488 conjugated goat anti-chicken was used as the secondary.

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Immunocytochemical/ Immunoflurescence analysis of cytospined HEK-293 cells (Human epithelial cell line from embryonic kidney) transfected with GFP, labeling GFP with ab13970 at 1/200 incubated for 16 hours at 4°C with 1% BSA in PBS. Secondary used was a donkey anti-chicken polyclonal DyLight^® 594 at 1/500.

GFP is shown in red (DyLight^® 594).

Nuclei are counterstained in blue (DAPI).

The left panel shows HEK-293 cells transfected with GFP and the right panel shows non-transfected HEK-293 cells.

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