All lanes : Anti-eIF4EBP1 antibody [Y329] (ab32024) at 1/2000 dilution (Purified)

Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 3 : Mouse skeletal muscle lysate
Lane 4 : Rat skeletal muscle lysate
Lane 5 : L6 (Rat skeletal muscle myoblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

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

All lanes : Anti-eIF4EBP1 antibody [Y329] (ab32024)

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : elF4EBP1 knockout HAP1 cell lysate
Lane 3 : HEK293 cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 13 kDa

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.

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

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).

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.

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.

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.

All lanes : Anti-eIF4EBP1 antibody [Y329] (ab32024) at 1/5000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : EIF4EBP1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 13 kDa
Observed band size: 13 kDa

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.

 

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.

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.