All lanes : Anti-N myc interactor/NMI antibody [EPR11065(2)] (ab183724) at 1/1000 dilution

Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : NMI knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : K562 (Human chronic myelogenous leukemia lymphoblast 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: 35 kDa
Observed band size: 39 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab183724 observed at 39 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab183724 Anti-N myc interactor/NMI antibody [EPR11065(2)]  was shown to specifically react with N myc interactor/NMI in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267013 (knockout cell lysate ab258077) was used. Wild-type and N myc interactor/NMI knockout samples were subjected to SDS-PAGE. ab183724 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.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling N myc interactor/NMI with ab183724 at 1/50 dilution. The slide is counterstained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: N myc interactor/NMI knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: K562 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab183724 observed at 39 kDa. Red - loading control, ab18058, observed at 124 kDa.
ab183724 was shown to specifically react with N myc interactor/NMI when N myc interactor/NMI knockout samples were used. Wild-type and N myc interactor/NMI knockout samples were subjected to SDS-PAGE. ab183724 and ab18058 (loading control to Vinculin) were diluted at 1/2000 and 1/10 000 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 hour at room temperature before imaging.

All lanes : Anti-N myc interactor/NMI antibody [EPR11065(2)] (ab183724) at 1/20000 dilution

Lane 1 : K562 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 35 kDa

Immunofluorescence analysis of acetone-fixed HeLa cells labeling N myc interactor/NMI with ab183724 at 1/100 dilution. Goat anti-rabbit IgG (Alexa Fluor® 488) at 1/200 dilution was used as the secondary antibody (green). The slide on the right is stained with Dapi (blue).

Western blot analysis of K562 cell lysate precipitated with ab183724 at 1/50 dilution. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution was then used. The blocking buffer and dilution buffer was 5% NFDM/TBST.