Anti-GFP Antibody (ab290) - Western Blot

Immunoprecipitation - Anti-GFP antibody (ab290)

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

Immunocytochemistry - Anti-GFP antibody (ab290)

GFP immunofluorescence with GFP antibody ab290, images showing similar localization of Yes-GFP (first 10 aa's of Yes PTK fused to the N-terminus of GFP) to full length Yes PTK. A: Distribution of Yes detected using mouse anti-Yes Ab followed by Texas Red-conjugated anti-mouse Ab. B: Chimeric GFP's detected using rabbit anti-GFP Ab (Abcam ab290) followed by FITC-conjugated anti-rabbit Ab.

Image kindly provided by L.G. Berthiaume. Taken from J. McCabe and L.G. Berthiaume, Functional Roles for Fatty Acylated Amino-terminal Domains in Subcellular Localization, Molecular Biology of the Cell 10:3771-3786, 1999

Immunocytochemistry - Anti-GFP antibody (ab290)

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).

Immunoprecipitation - Anti-GFP antibody (ab290)

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

Immunocytochemistry - Anti-GFP antibody (ab290)

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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody (ab290)

Bone marrow-derived infiltrating cells in the stromal tissue of gastric intraepithelial tumor traced by GFP direct fluorescence.

(A) Normal tissues of the glandular stomach of a regular GFP(−) control mouse. (B) Normal tissues of the glandular stomach of a GFP(+) transgenic control mouse; (C, E, D, F) An induced gastric intraepithelial neoplasia (GIN) in a bone marrow transplanted mouse. GFP(+) BMDCs tracked with direct fluorescence localized in the GIN stromal tissue are shown in C and E. The same GIN lesion slide stained by H&E after the fluorescence observation are shown in D and F. DAPI (A–C and E) and hematoxylin (D and F) are used to visualize nuclei, respectively. Locations of the images C and D in the images E and F, and the image E in the image F are marked in the corresponding color. The gastric glands and stromal cells are also labeled.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody (ab290)

Immunohistochemistry - Free Floating - 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).

Immunohistochemistry - Free Floating - Anti-GFP antibody (ab290)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody (ab290)

GFP staining with ab290 in dog hearts (Adv-GFP injection) tissue sections by IHC-P. Sections were PFA fixed and subjected to heat mediated antigen retrieval in citric acid (Ph6.0, 0.05% Tween20) prior to blocking with 10% serum for 30 mins at 37°C. The primary antibody was diluted 1/1000 in PBS and incubated with the sample for 1 hour at 25°C. A HRP conjugated secondary like Goat Anti-Rabbit IgG H&L (HRP) (ab205718) was used.