Anti-NNMT antibody [EPR29205-77]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNMT antibody [EPR29205-77] (AB318280)

Immunohistochemical analysis of paraffin-embedded A Wild-type Hela (Human cervix adenocarcinoma) cell pellet B Human NNMT knockout Hela cell pellet tissue labeling NNMT with ab318280 at 1/1000 (0.505 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) wild-type Hela cell pellet, no staining on (B) human NNMT knockout Hela cell pellet. The section was incubated with ab318280 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNMT antibody [EPR29205-77] (AB318280)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling NNMT with ab318280 at 1/2000 (0.253 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of human colon carcinoma. The section was incubated with ab318280 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNMT antibody [EPR29205-77] (AB318280)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling NNMT with ab318280 at 1/2000 (0.253 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stroma of human breast carcinoma. The section was incubated with ab318280 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNMT antibody [EPR29205-77] (AB318280)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling NNMT with ab318280 at 1/2000 (0.253 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human liver. The section was incubated with ab318280 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NNMT antibody [EPR29205-77] (AB318280)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling NNMT with ab318280 at 1/500 (1.01 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear and cytoplasmic staining in parental HeLa cell line (shown in green) and negative staining in NNMT KO HeLa cell line. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NNMT antibody [EPR29205-77] (AB318280)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling NNMT with ab318280 at 1/2000 (0.253 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on human cerebrum. The section was incubated with ab318280 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Supplier Data

Western blot - Anti-NNMT antibody [EPR29205-77] (AB318280)

Negative control : PANC-1.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

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

All lanes:

Western blot - Anti-NNMT antibody [EPR29205-77] (ab318280) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 2:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 29 kDa,36 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-NNMT antibody [EPR29205-77] (AB318280)

Negative control : cerebellum, skeletal muscle (PMID : 8182091).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

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

All lanes:

Western blot - Anti-NNMT antibody [EPR29205-77] (ab318280) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human cerebellum tissue lysate at 20 µg

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 29 kDa,36 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-NNMT antibody [EPR29205-77] (AB318280)

The samples were run on a Bis-Tris gel under reducing conditions.

Western blot : Anti-NNMT antibody (ab318280) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta.

In Western blot, ab318280 was shown to bind specifically to NNMT. Target of interest was observed at 29 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in NNMT knockout cell line (lane 2, knockout cell line ab265700 / knockout cell lysate ab258537). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

Negative control : MCF7 (PMID : 24558488).

All lanes:

Western blot - Anti-NNMT antibody [EPR29205-77] (ab318280) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

NNMT knockout HeLa whole cell lysate at 20 µg

Lane 3:

786-O (human kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 29 kDa

false

• WB

Supplier Data

Western blot - Anti-NNMT antibody [EPR29205-77] (AB318280)

This antibody does not cross-react with human INMT.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

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

All lanes:

Western blot - Anti-NNMT antibody [EPR29205-77] (ab318280) at 1/1000 dilution

Lane 1:

His-tagged human NNMT recombinant protein at 10 ng

Lane 2:

His-tagged human INMT recombinant protein at 10 ng

Secondary

All lanes:

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

Observed band size: 29 kDa

false

Exposure time: 10s