All lanes : Anti-NEK9 antibody [EP7361] (ab138488) at 1/1000 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : NEK9 knockout A431 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 107 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab138488 observed at 120 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab138488 was shown to react with NEK9 in wild-type A431 cells in western blot. Loss of signal was observed when NEK9 knockout sample was used. Wild-type and NEK9 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab138488 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling NEK9 with Purified ab138488 at 1:100 dilution (3.8μg/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). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified ab53281 at 1/20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - cells without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling NEK9 with Purified ab138488 at 1:100 dilution (3.84 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Anti-NEK9 antibody [EP7361] (ab138488) at 1000 cells (purified) + Rat spleen lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 107 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-NEK9 antibody [EP7361] (ab138488) at 1/5000 dilution (purified)

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 2 : THP-1 (Human monocytic leukemia monocyte) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 107 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling NEK9 with unpurified ab138488 at 1/50 dilution.

Immunofluorescent analysis of HeLa cells labelling NEK9 with unpurified ab138488 at 1/250 dilution.

All lanes : Anti-NEK9 antibody [EP7361] (ab138488) at 1/1000 dilution (unpurified)

Lane 1 : NIH3T3 cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : THP1 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 107 kDa