ab108382 staining CD13 in wild-type THP-1 cells (top panel) and ANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab108382 at 1/1000 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling CD13 with Purified ab108382 at 1:750 dilution (0.5 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). PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-CD13 antibody [EPR4059] (ab108382) at 1/1000 dilution

Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : ANPEP knockout THP-1 cell lysate
Lane 3 : PANC-1 cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 110 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab108382 observed at 160 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108382 was shown to react with CD13 in wild-type THP-1 cells in western blot with loss of signal observed in ANPEP knockout cell line ab273759 (knockout cell lysate ab275505). Wild-type and ANPEP knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab108382 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 A375 (human malignant melanoma epithelial cell) cells labeling CD13 with purified ab108382 at 1:500 (0.7 μg/ml). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with None. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 μg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ab108382 (purified) at 1:20 dilution (2ug) immunoprecipitating in THP-1 whole cell lysate. THP-1 (Human monocytic leukemia monocyte) whole cell lysate 10ug
Lane 2 (+): ab108382 & THP-1 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab108382 in THP-1 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-CD13 antibody [EPR4059] (ab108382) at 1/1000 dilution

Lane 1 : Rat lung lysates
Lane 2 : A375 (Human malignant melanoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 110 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling CD13 with purified ab108382 at 1:750 dilution (0.5 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). PBS instead of the primary antibody was used as the negative control.

Unpurified ab108382 staining CD13 in human kidney tissue sections by Immunohistochemistry (Formaldehyde/PFA-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde, permeabilized with 0.05% tween-20 and blocked for 60 minutes at 25°C. Antigen retrieval was by heat mediation. Samples were incubated with primary antibody at a dilution of 1/400 for 1 hour at 25°C. An Alexa Flour^® 488-conjugated donkey anti-rabbit IgG polyclonal (1/1200) was used as the secondary antibody.

See Abreview

All lanes : Anti-CD13 antibody [EPR4059] (ab108382) at 1/1000 dilution (unpurified)

Lane 1 : THP-1 cell lysate
Lane 2 : U937 cell lysate
Lane 3 : A375 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 110 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Immunohistochemical staining of paraffin-embedded Human kidney tissue using unpurified ab108382 at a dilution of 1/100.

Unpurified ab108382 showing positive staining in human Prostatic carcinoma tissue.

Unpurified ab108382 showing positive staining in human Normal liver tissue.

Unpurified ab108382 showing positive staining in human Ovarian carcinoma tissue.

Unpurified ab108382 showing positive staining in human Hepatocellular carcinoma tissue.

Unpurified ab108382 showing positive staining in Normal human breast tissue.

Unpurified ab108382 showing positive staining in Normal human tonsil tissue.