Recombinant Anti-NDRG1 antibody [EPR5593] (ab124689)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NDRG1 with purified ab124689 at 1/20 dilution (5 ug/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as a isotype control. Cell without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling NDRG1 using ab124689. The cells were fixed with 100% Methanol then permeabilized with 0.1% Triton X-100. The cells were then incubated with ab124689 at 1:50 dilution followed by a further incubation with a Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI. Cells were counterstained using ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1:200 dilution (shown in red). Secondary antibody only control: PBS instead of the primary antibody.

Immunohistochemical analysis of Paraffin-embedded sections mouse colon tissue labelling NDRG1 with ab124689 at 1/1000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on mouse colon tissue is observed. Counter stained with Haematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0).

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of Paraffin-embedded sections rat colon tissue labelling NDRG1 with ab124689 at 1/1000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on rat colon tissue is observed. Counter stained with Haematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0).

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of Paraffin-embedded sections human liver carcinoma tissue labelling NDRG1 with ab124689 at 1/1000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on human liver carcinoma tissue is observed. Counter stained with Haematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0).

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of Paraffin-embedded sections human colon tissue labelling NDRG1 with ab124689 at 1/1000 dilution, followed by a ready to use secondary Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Staining on human colon tissue is observed. Counter stained with Haematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0).

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

ab181602 was used as GAPDH loading control.

Lanes 1-4: Merged signal (red and green). Green - ab124689 observed at 43 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab124689 Anti-NDRG1 antibody [EPR5593]  was shown to specifically react with NDRG1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab267301 (knockout cell lysate ab257551) was used. Wild-type and NDRG1 knockout samples were subjected to SDS-PAGE. ab124689 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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.

Lanes 1 - 4: Merged signal (red and green). Green - ab124689 observed at 43 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab124689 was shown to recognize in wild-type HEK-293 cells as signal was lost at the expected MW in NDRG1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NDRG1 knockout samples were subjected to SDS-PAGE. Ab124689 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg.

Lane 2 (+): ab124689 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab124689 in HeLa whole cell lysate

ab124689 (Purified) at 1/50 dilution (20µg/ml) immunoprecipitating NDRG1 in HeLa whole cell lysate. For western blotting, ab124689 at 1/500 dilution (1.86 µg/mL) and VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1000 dilution.

Blocking and diluting buffer: 5% NFDM /TBST .