ab108310 staining CD13 in wild-type THP-1 cells (top panel) and ANPEP knockout THP-1 cells (bottom panel) (ab273759). The cells were fixed with 4% paraformaldehyde (10 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 ab108310 at 1/500 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 human kidney tissue sections labeling CD13 with purified ab108310 at 1/1600 dilution (0.43 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-CD13 antibody [EPR4058] (ab108310) 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 - ab108310 observed at 160 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108310 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 fluorescent western blot (TBS-based) blocking solution before incubation with ab108310 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.

Confocal image showing membranous staining in THP-1 cells

ab108310 (purified) at 1/100 staining CD13 in the THP-1 (human monocytic leukemia monocyte) cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 100% methanol. Samples were incubated with primary antibody 1/500. ab150077 An Alexa Fluor® 488-conjugated Goat anti-Rabbit IgG at 1/1000 was used as the secondary antibody. Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 was used as a counter stain and DAPI was used as a nuclear counter stain. 

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Empty
Lane 3: CD13 knockout HAP1 whole cell lysate (20 µg)
Lane 4: Empty
Lane 5: HeLa whole cell lysate (20 µg)

Lanes 1 - 5: Merged signal (red and green). Green - ab108310 (unpurified) observed at 160 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab108310 was shown to recognize CD13 when CD13 knockout samples were used, along with additional cross-reactive bands. Wild-type and CD13 knockout samples were subjected to SDS-PAGE.  Ab108310 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed 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 1/10000 dilution for 1 hour at room temperature before imaging.

Anti-CD13 antibody [EPR4058] (ab108310) at 1/20000 dilution (Purified) + Mouse kidney lysates at 15 µg

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

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

All lanes : Anti-CD13 antibody [EPR4058] (ab108310) at 1/5000 dilution (Purified)

Lane 1 : THP-1 (Human monocytic leukemia monocyte) whole cell lysates
Lane 2 : Rat kidney 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: 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 rat kidney tissue sections labeling CD13 with purified ab108310 at 1/1600 dilution (0.43 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) 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 kidney tissue sections labeling CD13 with purified ab108310 at 1/1600 dilution (0.43 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-CD13 antibody [EPR4058] (ab108310) at 1/1000 dilution (unpurified)

Lane 1 : THP-1 cell lysate at 10 µg
Lane 2 : human fetal liver lysate

Performed under reducing conditions.

Predicted band size: 110 kDa

ab108310 (unpurified), at 1/250, staining CD13 in human kidney tissue by immunohistochemistry. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human normal liver tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human hepatocellular carcinoma tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human astrocytoma tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human normal breast tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human prostatic carcinoma tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human normal tonsil tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human normal stomach tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human normal colon tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

ab108310 (unpurified) showing positive staining in human lung adenocarcinoma tissue. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.