Anti-EIF2S1 antibody [EIF2a]

• WB

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Western blot - Anti-EIF2S1 antibody [EIF2a] (AB5369)

All lanes:

Western blot - 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, 2, 3, 4, 6 and 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

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EIF2S1 antibody [EIF2a] (AB5369)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EIF2S1 antibody [EIF2a] (AB5369)

Immunohistochemistry analysis of EIF 2ALPHA (EIF2A) showing staining in the cytoplasm of paraffin-embedded human colon carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab5369.

• ICC

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Immunocytochemistry - Anti-EIF2S1 antibody [EIF2a] (AB5369)

Immunofluorescence analysis of eIF2a was done on 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ab5369 1 : 250 dilution in1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1 : 2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin (1 : 300). Panel d is a merged image showing Cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

• ICC

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Immunocytochemistry - Anti-EIF2S1 antibody [EIF2a] (AB5369)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EIF2S1 antibody [EIF2a] (AB5369)

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