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

The antibody was used to detect transplanted bone marrow derived cells in paraffin embedded mouse brain tissue. The image was taken at 40X magnification. The GFP290 antibody(red) was visualized with a Cy3 anti-rabbit (Jackson Immuno) and the nuclei (blue) have been counterstained with bisbenzimide (Hoechst Stain). The image illustrates vascular association. The picture was kindly given to Abcam by the authors of the reference Hess, D.C et al. Bone Marrow as a Source of Endothelial Cells and NeuN-Expressing Cells After Stroke. Stroke 33(5) pp 1362-1368 (2002).

Immunohistochemistry (Frozen sections) - GFP antibody (ab290)

ab290 at a 1/2000 dilution staining GFP-labelled nerve fibres from Axolotls (Ambystoma mexicanum). The tissue sections were paraformaldehyde fixed and blocked with serum prior to incubation with the antibody for 24 hours. Bound antibody was detected using a biotinylated goat anti-rabbit polyclonal antibody.

This image is courtesy of an Abreview submitted by Miss Danielle Harlow

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

ab290 staining 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 goat anti-rabbit IgG was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Anti-GFP antibody (ab290)

ab290 staining GFP in human HEK293 cells by Immunocytochemistry/ Imunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton ×100 in PBS and blocking in 3% BSA and 2% Goat Serum was performed at 22⁰C for 1 hour. Samples were incubated with primary antibody (1/1000: in 3% BSA, 2% Goat Serum in 0.1%Triton X100 PBS) for 12 hours at 4°C. An Alexa Fluor^®555-conjugated goat polyclonal to rabbit IgG (H&L) was used as secondary antibody. In the figure, ab290 antibody specifically binds to the HEK293 cells expressing GFP. The non-GFP-expressing cells are not recognized by ab290 (pointed with white arrow).

This image is a courtesy of Anonymous Abreview

Western blot - GFP antibody (ab290)

Anti-GFP antibody (ab290) at 1/10000 dilution + Lysate prepared from rabbit reticulocytes at 3 µg

Secondary
HRP-conjugated donkey monoclonal to rabbit IgG at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Observed band size : 27 kDa (why is the actual band size different from the predicted?)
Additional bands at : 60 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 5 seconds

This image is a courtesy of Anonymous Abreview

Western blot - GFP antibody (ab290)

All lanes : Anti-GFP antibody (ab290) at 1/30000 dilution

Lane 1 : GFP tagged protein transfected HEK293 cell lysates
Lane 2 : GFP protein alone

Lysates/proteins at 25 µg per lane.

Secondary
HRP-conjugated Goat polyclonal to rabbit IgG at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Observed band size : 24,64 kDa (why is the actual band size different from the predicted?)


Exposure time : 3 minutes

Primary diluted in 1XPBST, 5% milk and incubated with sample for 16 hour at 4°C.

The image is a courtesy of an abreview submitted by Vladimir Milenkovic.

Immunoprecipitation - GFP antibody (ab290)

ab290 Immunoprecipitate in human HEK 293 cells transfected with Annexin1-GFP. 25µg of cell lysate incubated with primary antibody and matrix (Protein G) in 1% TX-100, 10% glycerol, 1X PBS for 16 hours at 4ºC. For Western blotting an HRP conjugated HRP goat polyclonal to rabbit Ig was used at a dilution at 1/5000. Line 1: Lysate of HEK 293 cells expressing Annexin1-GFP fusion Line 2: IP with anti GFP Ab Line 3: Not bound fraction

This image is courtesy of an Abreview submitted by Vladimir Milenkovic

Western blot

Anti-GFP antibody (ab290) at 1/2500 dilution + GFP protein (Active) (ab84191) at 0.01 µg

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 30 seconds

Immunocytochemistry/ Immunofluorescence - GFP antibody (ab290)

Immunofluorescence 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