All lanes : Anti-EIF2S1 antibody [EIF2a] (ab5369)

Lane 1 : COS-7 (african green monkey kidney fibroblast-like cell line) whole cell lysate at 20 µg
Lane 2 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 20 µg
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 5 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 6 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate
Lane 7 : A431 (human epidermoid carcinoma cell line) whole cell lysate

Secondary
Lanes 1-4 & 6-7 : Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate at 1/4000 dilution
Lane 5 : Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate

Predicted band size: 36 kDa

Immunohistochemical analysis of human colon carcinoma (right) compared to a negative control (left) using ab5369 at the dilution 1/10.

ICC/IF image of ab5369 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab5369, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Ab5369 staining EIF2S1 in human placenta. Staining is localised to the cytoplasm.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.