Anti-GFP antibody(AB290)

GFP Immunohistochemistry (Free Floating) analysis of mouse brain tissue sections with ab290. Tissue was fixed with 4% PFA frozen 30 μm sections were blocked for 1 hour at room temperature with 10% normal goat serum + donkey anti-mouse IgG Fab fragments (0.1 mg/ml). Sections were incubated with the primary antibody at a dilution of 1/1000 in TBS + 0.25% Triton-X for 16 hours at 4°C. A Cy2®-conjugated donkey anti-rabbit IgG (H+L) at a dilution of 1/200 was used as the secondary antibody.

Image shows anti-NeuN (red) DAPI (blue) and anti-GFP staining of GFP-cre (green yellow with NeuN colocalization).

GFP staining with GFP antibody ab290 in U2OS cells expressing TRF2-GFP fusion protein by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde permeabilized with NP40 and blocked with 3% BSA for 1 hour at 21°C. Samples were incubated with the primary antibody (1/1000 in PBS + 3% BSA) for 12 hours at 4°C. An Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at a dilution of 1/500 was used as the secondary antibody.

Green - GFP.

Blue - DAPI.

GFP immunoprecipitation with GFP antibody ab290 in human HEK293 cells transfected with Annexin1-GFP. 25μg of cell lysate was incubated with the primary antibody and matrix (Protein G) in 1% TX-100, 10% glycerol, 1X PBS for 16 hours at 4°C. For Western blotting anti-rabbit HRP conjugated secondary antibody was used at a dilution at 1/5000.

Lane 1 : Lysate of HEK293 cells expressing Annexin1-GFP fusion protein.

Lane 2 : IP with anti-GFP.

Lane 3 : Not bound fraction.

All lanes:

Immunoprecipitation - Anti-GFP antibody (ab290)

Predicted band size: 27 kDa

This image is courtesy of an Abreview submitted by Vladimir Milenkovic

GFP immunoprecipitation with ab290 in HEK293 nuclear lysate expressing GFP. 20μg of lysate was incubated with primary antibody (1 μg/mg lysate) and matrix (Protein G) for 16 hours at 4°C in AFC low salt buffer. For western blotting ab290 (1/5000) was used to confirm successful immunoprecipation.

Lane 1 : HEK293 nuclear lysate expressing GFP input.

Lane 2 : IP of HEK293 nuclear lysate expressing GFP.

Lane 3 : Cells with no GFP.

All lanes:

Immunoprecipitation - Anti-GFP antibody (ab290)

Predicted band size: 27 kDa

This image is courtesy of an Abreview submitted by William Hung

GFP staining with GFP antibody ab290 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 ab290 at 1/200 dilution overnight at +4°C followed by incubation with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) 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 ab290 was applied gave a stronger signal in the 488 channel indicating that ab290 is binding to GFP and therefore eliciting signal amplification.

ab290 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).