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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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).

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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