Anti-DDAH1 antibody [EPR13922]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-DDAH1 antibody [EPR13922] (AB180599)

ab180599 staining DDAH1 in the human cell line 293 (human embryonic kidney) by intracellular 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)

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-DDAH1 antibody [EPR13922] (AB180599)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDAH1 antibody [EPR13922] (AB180599)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-DDAH1 antibody [EPR13922] (AB180599)

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.

All lanes:

Immunoprecipitation - Anti-DDAH1 antibody [EPR13922] (ab180599)

Predicted band size: 31 kDa

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• WB

Lab

Western blot - Anti-DDAH1 antibody [EPR13922] (AB180599)

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:

Western blot - Anti-DDAH1 antibody [EPR13922] (ab180599) at 1/10000 dilution

Lane 1:

Wild-type A431 cell lysate at 40 µg

Lane 2:

Western blot - Human DDAH1 knockout A-431 cell lysate (<a href='/en-us/products/cell-lysates/human-ddah1-knockout-a-431-cell-lysate-ab261715'>ab261715</a>) at 40 µg

Lane 3:

HUVEC (Human umbilical vein endothelial cell line) whole cell lysate at 40 µg

Lane 4:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 40 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 31 kDa

Observed band size: 37 kDa,55 kDa

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• WB

Supplier Data

Western blot - Anti-DDAH1 antibody [EPR13922] (AB180599)

All lanes:

Western blot - 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

Predicted band size: 31 kDa

Observed band size: 37 kDa

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