All lanes : Anti-GFP antibody [LGB-1] (ab291) at 0.5 µg/ml

Lane 1 : 5ng GFP
Lane 2 : 10ng GFP
Lane 3 : 25ng GFP

Secondary
All lanes : Sheep anti-mouse IgG HRP conjugate at 1/5000 dilution

Predicted band size: 27 kDa
Observed band size: 27 kDa

Paraformaldehyde fixed COS-7 cells expressing Myr-N15-PAK2-EGFP construct (Vilas et al.(2006) PNAS 103, 6542). Myr-N15-PAK2-EGFP fluorescence is shown in green. Indirect immunofluorescenct detection of N15-PAK-EGFP using ab291 monoclonal LGB-1 anti-GFP at 0.05 ug/ml with chicken anti-mouse secondary antibody conjugated to Alexa594 diluted 1/500 is shown in red. Myr-N15-PAK2-EGFP is localized to membrane ruffles and perinuclear vesicular structures (likely Golgi,TGN or late endosomes).

ab291 at 6.7µg/mg lysate.
HEK293 Cell lysate at 300µg.Transfected with CFP-fused protein XXX in pECFP vector. Immunoprecipitation step using Protein G.

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ab291 staining GFP in Dog MDCKII cells transfected with GFP by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% TX100 and blocked with 5% serum for 20 minutes. Samples were incubated with primary antibody (1/250 PBS + 0.1% TX100 + 1% goat serum) for 16 hours at 4°C. An Alexa Fluor^®546-conjugated Goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody. DAPI was used to stain nuclei. ab291 was used to assess electoporation efficiency of double transfected MDCKII cells.

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ab291 staining GFP in GFP-transfected NIH3T3 cells. The cells were fixed with 4% formaldehyde (10min) and then blocked in 1% BSA / 0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab291 at 1/200 dilution overnight at +4°C followed by incubation with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488), for 1 hour, at 1μg/ml.
Under identical experimental conditions, when compared to the basal level of GFP expression in transfected NIH3T3 cells, the cells upon which ab291 was applied gave a stronger signal in the 488 channel, indicating that ab291 is binding to GFP and therefore eliciting signal amplification.
ab291 was also applied to non-GFP-transfected NIH3T3 cells, which produced no positive staining, indicating specificity for GFP. Nuclear DNA was labelled with 1.43μM DAPI (blue).