All lanes : Anti-ASS1 antibody [EPR12398] (ab170952) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ASS1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 47 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab170952 observed at 47 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab170952 was shown to react with ASS1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264989 (knockout cell lysate ab257143) was used. Wild-type HeLa and ASS1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab170952 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab170952 staining ASS1 in wild-type HeLa cells (top panel) and ASS1 knockout HeLa cells (ab264989) (bottom panel). 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 ab170952 at 1/100 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).

All lanes : Anti-ASS1 antibody [EPR12398] (ab170952) at 1/20000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ASS1 knockout HAP1 whole cell lysate
Lane 3 : HepG2 whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 47 kDa
Observed band size: 47 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab170952 observed at 47 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab170952 was shown to recognize ASS1 in wild-type HAP1 cells as signal was lost at the expected MW in ASS1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and ASS1 knockout samples were subjected to SDS-PAGE. Ab170952 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/20000 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.

All lanes : Anti-ASS1 antibody [EPR12398] (ab170952) at 0.05 µg/ml (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : Human fetal liver lysate
Lane 3 : Mouse liver lysate
Lane 4 : Rat liver lysate
Lane 5 : Mouse kidney lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 47 kDa
Observed band size: 47 kDa

Blocking and diluting buffer: 5% NFDM/TBST

ab170952 (purified) at 1:60 dilution (5ug) immunoprecipitating ASS1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab170952 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab170952 in HeLa (Human cervix adenocarcinoma epithelial cell) 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.

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling ASS1 with Purified ab170952 at 1:100 dilution (10.2μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ab170952 showing +ve staining in Mouse kidney tissue.

Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ASS1 with purified ab170952 at 1:100 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunofluorescent analysis of HeLa cells labeling ASS1 using ab170952 at 1/50 dilution (red). DAPI nuclear staining (blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse kidney tissue sections labeling ASS1 with Purified ab170952 at 1:4000 dilution (0.25 μg/ml). Heat mediated antigen retrieval was performed using citrate Buffer, PH6. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling ASS1 with Purified ab170952 at 1:4000 dilution (0.25 μg/ml). Heat mediated antigen retrieval was performed using citrate Buffer, PH6. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

ab170952 showing +ve staining in Human normal ureter tissue.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling ASS1 with ab170952 at 1/250 dilution.

ab170952 showing -ve staining in Human cervical carcinoma tissue.

ab170952 showing -ve staining in Human normal uterus tissue.

ab170952 showing -ve staining in Human normal pancreas tissue.

Secondary antibody used is HRP-conjugated anti-rabbit IgG preferentially detecting the non-reduced form of rabbit IgG.

All lanes : Anti-ASS1 antibody [EPR12398] (ab170952) at 1/10 dilution

Lane 1 : Human fetal liver tissue lysate at 10 µg
Lane 2 : PBS

Flow cytometric analysis of permeabilized Hela cells labeling ASS1 using ab170952 at 1/500 dilution  (red) or a rabbit IgG negative (green).

All lanes : Anti-ASS1 antibody [EPR12398] (ab170952) at 1/1000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : Fetal liver tissue lysate
Lane 3 : Fetal kidney tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 47 kDa