Anti-eIF4EBP1 antibody [Y329]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-EIF4EBP1 knockout cells (red line) stained with ab32024. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32024, 0.1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-EIF4EBP1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min) , permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Intracellular Flow Cytometry analysis of HEK-293 (Human embryonic kidney epithelial cell) cells labeling eIF4EBP1 with purified ab32024 at 1/20 dilution (10 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 (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling eIF4EBP1 with Purified ab32024 at 1/500 dilution (5 μg/ml). Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) at 1/1000 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon cancer tissue sections labeling eIF4EBP1 with purified ab32024 at 1/200 dilution (0.53 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). 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-eIF4EBP1 antibody [Y329] (AB32024)

Purified ab32024 at 1/20 dilution (2μg) immunoprecipitating eIF4EBP1 in HEK-293 whole cell lysate.
Lane 1 (input) : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32024 + HEK-293 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32024 in HEK-293 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 15-20 kDa

All lanes:

Immunoprecipitation - Anti-eIF4EBP1 antibody [Y329] (ab32024)

Predicted band size: 13 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue sections labeling eIF4EBP1 with purified ab32024 at 1/200 dilution (0.53 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue sections labeling eIF4EBP1 with purified ab32024 at 1/200 dilution (0.53 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). 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

Unknown

Western blot - Anti-eIF4EBP1 antibody [Y329] (AB32024)

The molecular weights observed are consistent with what has been described in the literatures (PMID : 28613975, 20890458 and 27358481).

All lanes:

Western blot - Anti-eIF4EBP1 antibody [Y329] (ab32024) at 1/2000 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 3:

Mouse skeletal muscle lysate at 20 µg

Lane 4:

Rat skeletal muscle lysate at 20 µg

Lane 5:

L6 (Rat skeletal muscle myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 13 kDa

Observed band size: 18 kDa

false

• WB

Lab

Western blot - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32024 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32024 was shown to specifically react with elF4EBP1 in wild-type Hap1 cells. Wild-type and elF4EBP1 knockout samples were subjected to SDS-PAGE. ab32024 and ab8245 (loading control to GADPH) were diluted 1/5000 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 [Y329] (ab32024)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

elF4EBP1 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

false

• WB

Lab

Western blot - Anti-eIF4EBP1 antibody [Y329] (AB32024)

Lanes 1-2 : Merged signal (red and green). Green - ab32024 observed at 13 kDa. Red - loading control ab8245 observed at 37 kDa.

ab32024 Anti-eIF4EBP1 antibody [Y329] 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. ab32024 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 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 [Y329] (ab32024) at 1/5000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human EIF4EBP1 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-eif4ebp1-knockout-hela-cell-lysate-ab257146'>ab257146</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 13 kDa

Observed band size: 13 kDa,37 kDa

false