All lanes : Anti-ULK3 antibody [EPR4888] (ab124947) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : ULK3 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HAP1 whole cell lyate
Lane 4 : LNCaP (Human prostate cancer cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 53 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab124947 observed at 55 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab124947 Anti-ULK3 antibody [EPR4888] was shown to specifically react with ULK3 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266152 (knockout cell lysate ab258271) was used. Wild-type and ULK3 knockout samples were subjected to SDS-PAGE. ab124947 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue sections labeling ULK3 with purified ab124947 at 1/50 dilution (7.84 µg/mL). Heat mediated antigen retrieval was performed 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.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunocytochemistry/Immunofluorescence analysis of LNCaP (Human prostate carcinoma epithelial cell) cells labeling ULK3 with Purified ab124947 at 1:50 dilution (7.8 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. 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 (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: ULK3 knockout HAP1 whole cell lysate (20 µg)
Lane 3: Hek293 whole cell lysate (20 µg)
Lanes 1 - 3: Merged signal (red and green). Green - ab124947 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124947 (unpurified) was shown to specifically react with ULK3 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when ULK3 knockout samples were examined. Wild-type and ULK3 knockout samples were subjected to SDS-PAGE. Ab124947 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-ULK3 antibody [EPR4888] (ab124947) at 1/1000 dilution (Purified)

Lane 1 : Human heart lysate
Lane 2 : Human testis lysate
Lane 3 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate
Lane 4 : HEK-293 (Human embryonic kidney epithelial cell) 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: 53 kDa
Observed band size: 53 kDa

Blocking/Diluting buffer: 5% NFDM/TBST

Flow Cytometry analysis of LNCaP (Human prostate carcinoma epithelial cell) cells, labeling ULK3 with Purified ab124947 at 1:40 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).

ab124947 (unpurified), at a 1/50 dilution, staining ULK3 in paraffin embedded Human bladder carcinoma tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.