Anti-eIF4EBP1 antibody [Y330]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-eIF4EBP1 antibody [Y330] (AB32130)

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling eIF4EBP1 with purified ab32130 at 1/1000 dilution (1 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Left).
Low expression in mitotic phase was consistent with literature. (PMID : 11553333)

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4EBP1 antibody [Y330] (AB32130)

ab32130, staining human prostate carcinoma by immunohistochemistry, Paraffin embedded tissue

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-eIF4EBP1 antibody [Y330] (AB32130)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32130 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32130 was shown to specifically react with eIF4EBP1 when eIF4EBP1 knockout samples were used. Wild-type and eIF4EBP1 knockout samples were subjected to SDS-PAGE. ab32130 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 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.

All lanes:

Western blot - Anti-eIF4EBP1 antibody [Y330] (ab32130) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

eIF4EBP1 knockout HAP1 cell lysate at 20 µg

Lane 3:

Hek293 cell lysate at 20 µg

Lane 4:

K562 cell lysate at 20 µg

Predicted band size: 13 kDa

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• WB

Unknown

Western blot - Anti-eIF4EBP1 antibody [Y330] (AB32130)

All lanes:

Western blot - Anti-eIF4EBP1 antibody [Y330] (ab32130) at 1/1000 dilution

All lanes:

K562 cell lysate

Predicted band size: 13 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-eIF4EBP1 antibody [Y330] (AB32130)

Lanes 1-2 : Merged signal (red and green). Green - ab32130 observed at 13 kDa. Red - loading control ab8245 observed at 37 kDa.

ab32130 Anti-eIF4EBP1 antibody [Y330] was shown to specifically react with eIF4EBP1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264784 (knockout cell lysate ab257146) was used. Wild-type and eIF4EBP1 knockout samples were subjected to SDS-PAGE. ab32130 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 Dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-eIF4EBP1 antibody [Y330] (ab32130) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

EIF4EBP1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human EIF4EBP1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-eif4ebp1-knockout-hela-cell-line-ab264784'>ab264784</a>)

Predicted band size: 13 kDa,36 kDa

Observed band size: 13 kDa

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