Anti-eIF4E antibody [Y448]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-eIF4E antibody [Y448] (AB33766)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4E antibody [Y448] (AB33766)

This image shows human breast carcinoma stained with ab33766

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-eIF4E antibody [Y448] (AB33766)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-eIF4E antibody [Y448] (AB33766)

ICC/IF image of ab33766 stained MCF7 cells. The cells were 4% formaldehyde (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 (ab33766, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4E antibody [Y448] (AB33766)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cervical carcinoma tissue sections labeling eIF4E with Purified ab33766 at 1 : 100 dilution (1.33 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101)was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

Unknown

Immunoprecipitation - Anti-eIF4E antibody [Y448] (AB33766)

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.

All lanes:

Immunoprecipitation - Anti-eIF4E antibody [Y448] (ab33766)

Predicted band size: 25 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4E antibody [Y448] (AB33766)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat kidney tissue sections labeling eIF4E with Purified ab33766 at 1 : 100 dilution (1.33 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101)was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4E antibody [Y448] (AB33766)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse stomach tissue sections labeling eIF4E with Purified ab33766 at 1 : 100 dilution (1.33 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101)was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Lab

Western blot - Anti-eIF4E antibody [Y448] (AB33766)

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

All lanes:

Western blot - Anti-eIF4E antibody [Y448] (ab33766) at 1/5000 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 25 kDa

Observed band size: 25 kDa

false

• WB

Unknown

Western blot - Anti-eIF4E antibody [Y448] (AB33766)

All lanes:

Western blot - Anti-eIF4E antibody [Y448] (ab33766) at 1/500 dilution

All lanes:

293 cell lysate

Predicted band size: 25 kDa

Observed band size: 30 kDa

false

• WB

Lab

Western blot - Anti-eIF4E antibody [Y448] (AB33766)

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

All lanes:

Western blot - Anti-eIF4E antibody [Y448] (ab33766) at 1/1000 dilution

All lanes:

Human brain lysates at 15 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 25 kDa

Observed band size: 25 kDa

false

• WB

Lab

Western blot - Anti-eIF4E antibody [Y448] (AB33766)

Blocking and diluting buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-eIF4E antibody [Y448] (ab33766) at 0.025 µg/mL

Lane 1:

Raw264.7 (Mouse abelson murine leukemia virus-induced tumor) whole cell lysate at 20 µg

Lane 2:

C6 (Rat glioma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 25 kDa

false