Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: eIF2A knockout HAP1 cell lysate (20 µg)
Lane 3: U87-MG cell lysate (20 µg)
Lane 4: MOLT4 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab157478 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab157478 was shown to recognize eIF2A when eIF2A knockout samples were used, along with additional cross-reactive bands. Wild-type and eIF2A knockout samples were subjected to SDS-PAGE. ab157478 and ab8245 (loading control to GAPDH) were diluted at 1/500 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

ab157478 staining eIF2A in wild-type HAP1 cells (top panel) and EIF2A knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol for 5 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab157478 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

 

All lanes : Anti-eIF2A antibody [EPR11041] (ab157478) at 1/1000 dilution

Lane 1 : Ramos cell lysate
Lane 2 : MOLT4 cell lysate
Lane 3 : U87-MG cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 65 kDa

Immunofluorescent analysis of Raji cells labeling eIF2A with ab157478 at 1/100 dilution.