ab227663 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 ab227663 at 1μg/ml concentration 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).

Formalin-fixed, paraffin-embedded human kidney tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

All lanes : Anti-CD13 antibody [SP187] (ab227663) at 1/400 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: 109 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 - ab227663 observed at 160 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab227663 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 ab227663 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 400 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 THP-1 (human monocytic leukemia monocyte) cells labeling CD13 with purified ab227663 at 1/25 (5.32µg/ml). Cells were fixed in 100% Methanol. 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 was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Anti-CD13 antibody [SP187] (ab227663) at 1/400 dilution + U937 (human histiocytic lymphoma cell line) cell lysate

Predicted band size: 109 kDa

Formalin-fixed, paraffin-embedded human tonsil tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human spleen tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human reactive lymph node tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human thymus tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human lung tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human liver tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human placenta tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human bladder tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human HK lymphoma tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human B-cell lymphoma tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human bone marrow tissue stained for CD13 using ab227663 at 1/100 dilution in immunohistochemical analysis.