All lanes : Anti-eIF4E antibody [Y449] (ab33768) at 1/1000 dilution

Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 4 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 5 : C6 (Rat glial tumor glial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 25 kDa
Observed band size: 28 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time:
Lane 1: 20 seconds.
Lanes 2-5: 8 seconds.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian cancer tissue labelling eIF4E with ab33768 at a dilution of 1/500. Antigen retrieval was performed ab93684, Tris/EDTA buffer, pH 9. A ready to use goat anti-rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Counter stained with Hematoxylin.

Immunocytochemistry/ Immunofluorescence analysis of RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling eIF4E with Purified ab33768 at 1/50 (10 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of HEK-293 (Human embryonickidney epithelial cell) cells labeling eIF4E with Purified ab33768 at 1/50 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

eIF4E was immunoprecipitated from 10 µg NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate with ab33768 at a 1/30 dilution. Western blot was performed from the immunoprecipitate using ab33768 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10 µg (Input).
Lane 2: ab33768 IP in NIH/3T3 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab33768 in NIH/3T3 whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat testis tissue labelling eIF4E with ab33768 at a dilution of 1/500. Antigen retrieval was performed ab93684, Tris/EDTA buffer, pH 9. A ready to use goat anti-rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Counter stained with Hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue labelling eIF4E with ab33768 at a dilution of 1/500. Antigen retrieval was performed ab93684, Tris/EDTA buffer, pH 9. A ready to use goat anti-rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Counter stained with Hematoxylin.