Recombinant Anti-eIF4E antibody [Y448] (ab33766)

Immunocytochemistry/ Immunofluorescence analysis of RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling eIF4E with Purified ab33766 at 1:500 dilution (0.3 µg/ml). Cells were fixed in 100% Methanol and permeabilized with None. 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 nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Intracellular Flow Cytometry analysis of HEK-293 (Human embryonic kidney epithelial cell) cells labeling eIF4E with Purified ab33766 at 1/200 dilution (1µg/ml) (red). Cells were fixed with 80% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 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). 

ab33766 (purified) at 1:20 dilution (0.6µg) immunoprecipitating eIF4E in HEK-293 whole cell lysate.
Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab33766 & HEK-293 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab33766 in HEK-293 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:10,000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Blocking and dilutin buffer and concentration: 5% NFDM/TBST

Blocking and dilutin buffer and concentration: 5% NFDM/TBST

Blocking and diluting buffer: 5% NFDM/TBST. 

Exposure time - Lane 1: 160 seconds
                         Lane 2: 70 seconds 

Overlay histogram showing HEK293 cells stained with ab33766 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab33766, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.