Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation. 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This IHC data was generated using the same anti-NDRG1 antibody clone, EPR5593, in a different buffer formulation (cat# ab124689).

ab124689 at 1/100 dilution, staining NDRG1 in Formalin fixed Paraffin-embedded Human colon tissue by Immunohistochemistry.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This ICC/IF data was generated using the same anti-NDRG1 antibody clone, EPR5593, in a different buffer formulation (cat# ab124689).

ab124689, at 1/100 dilution, staining NDRG1 in HeLa cells by Immunofluorescence.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

ab124689 showing positive staining in Normal kidney tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124689).

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation. 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation. 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation. 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

Clone EPR5593 (ab216457) has been successfully conjugated by Abcam. This image was generated using Anti-NDRG1 antibody [EPR5593] (Alexa Fluor® 647). Please refer to ab199471 for protocol details.

ab199471 staining NDRG1 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), 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 overnight at +4°C with ab at a 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at a 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

Clone EPR5593 (ab216457) has been successfully conjugated by Abcam. This image was generated using Anti-NDRG1 antibody [EPR5593] (Alexa Fluor® 488). Please refer to ab199233 for protocol details.

ab199233 staining NDRG1 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), 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 overnight at +4°C with ab199233 at 1/500 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

Lab

Immunoprecipitation - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

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 : 500 dilution (1.86 μg/ml) immunoprecipitating NDRG1 in HeLa whole cell lysate. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution.

Blocking and diluting buffer : 5% NFDM /TBST .

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124689).

All lanes:

Immunoprecipitation - Anti-NDRG1 antibody [EPR5593] (<a href='/en-us/products/primary-antibodies/ndrg1-antibody-epr5593-ab124689'>ab124689</a>)

Predicted band size: 43 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation. 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation. 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.

• WB

Lab

Western blot - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation.

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.

All lanes:

Western blot - Anti-NDRG1 antibody [EPR5593] (<a href='/en-us/products/primary-antibodies/ndrg1-antibody-epr5593-ab124689'>ab124689</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 2:

NDRG1 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 2:

Western blot - Human NDRG1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-ndrg1-knockout-hek-293t-cell-line-ab267301'>ab267301</a>)

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Secondary

All lanes:

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 1/10000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

Lab

Western blot - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using ab124689, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as GAPDH loading control.

All lanes:

Western blot - Anti-NDRG1 antibody [EPR5593] (<a href='/en-us/products/primary-antibodies/ndrg1-antibody-epr5593-ab124689'>ab124689</a>) at 1/10000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Mouse brain lysate at 20 µg

Lane 3:

Rat brain lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-NDRG1 antibody [EPR5593] - Low endotoxin, Azide free (AB216457)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124689).

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.

Lanes 1 - 4:

Western blot - Anti-NDRG1 antibody [EPR5593] (<a href='/en-us/products/primary-antibodies/ndrg1-antibody-epr5593-ab124689'>ab124689</a>) at 1/10000 dilution

Lane 1:

Western blot - Alexa Fluor® 647 Anti-NDRG1 antibody [EPR5593] (<a href='/en-us/products/primary-antibodies/alexa-fluor-647-ndrg1-antibody-epr5593-ab199471'>ab199471</a>)

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

NDRG1 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human NDRG1 knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-ndrg1-knockout-hek-293-cell-line-ab261856'>ab261856</a>)

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 4:

LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg

Predicted band size: 43 kDa

false