All lanes : Anti-DDAH1 antibody [EPR13922] (ab180599) at 1/10000 dilution

Lane 1 : A431 wild-type cell lysate
Lane 2 : A431 DDAH1 knockout cell lysate
Lane 3 : HuVEC cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Observed band size: 37 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab180599 observed at 37 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab180599 was shown to react with DDAH1 in A431 wild-type cells in Western blot. Loss of signal was observed when DDAH1 knockout sample was used. A431 wild-type and DDAH1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab180599 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-DDAH1 antibody [EPR13922] (ab180599) at 1/20000 dilution

Lane 1 : Human fetal brain lysate at 20 µg
Lane 2 : Human fetal liver lysate at 20 µg
Lane 3 : Human fetal kidney lysate at 20 µg
Lane 4 : 293 lysate at 10 µg

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

Observed band size: 37 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin embedded Human ovarian carcinoma tissue labeling DDAH1 with ab180599 at a 1/50 dilution. Prediluted HRP Polymer for Rabbit IgG secondary used. Counterstained with Hematoxylin.

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

ab180599 staining DDAH1 in the human cell line 293 (human embryonic kidney) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

Immunofluorescence analysis of 293 cells (fixative 4% paraformaldehyde) labeling DDAH1 with ab180599 at a 1/100 dilution (left image), and counterstained with Dapi (right image). Goat anti rabbit IgG (Dylight 555) secondary used at a 1/200 diution.

Immunoprecipitation analysis of Human fetal kidney lysate labeling DDAH1 with ab180599 at a 1/40 dilution. Goat Anti-Rabbit IgG (H+L) Peroxidase conjugated secondary used at a 1/1000 dilution.