Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Wildtype HeLa(human cervical adenocarcinoma epithelial cell) untreated Wildtype HeLa starvation 2 hours then treated with 200 mM PMA for 30 min NR4A2 KO HeLa(NR4A2 knockout HeLa) untreated NR4A2 KO HeLa starvation 2 hours then treated with 200 mM PMA for 30 min cells labelling Nurr1 with ab327018 at 1/200 (2.505 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased nuclear staining in wildtype HeLa cells (shown in green) following 2 hours of starvation and 30 min of 200 mM PMA treatment. No staining is observed in untreated wildtype cells or in NR4A2 knockout HeLa cells under the same conditions. 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).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 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 dilution.

• IP

Lab

Immunoprecipitation - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

Nurr1 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate with ab327018 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab327018 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate
Lane 2 : ab327018 IP in HeLa (human cervical adenocarcinoma epithelial cell) was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab327018 in HeLa was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate

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

Exposure time : 50 seconds.

All lanes:

Immunoprecipitation - Anti-Nurr1 antibody [EPR26338-73] (<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a> at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a> in HeLa was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 60 kDa,67 kDa

false

Exposure time: 50s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse brain tissue labeling Nurr1 with ab327018 at 1/1000 (0.501 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum.

The primary antibody was incubated for 30 mins at room temperature. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (fresh frozen) tissue labeling Nurr1 with ab327018 at 1/50 (10.02 μg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 μg/mL dilution (Green).

Panel A : merged staining of anti-NR4A2 (ab327018, green), anti-NeuN (ab190565, magenta) on mouse cerebrum. Panel B : anti-NR4A2 stained on mouse cerebrum. Panel C : anti-NeuN stained in neurons of mouse cerebrum. The section was incubated in two rounds of staining : in the order of ab327018 and ab190565, for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

• WB

Supplier Data

Western blot - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

ab327018 was shown to react with NR4A2 in wild-type HeLa cells in Western blot with loss of signal observed in a NR4A2 knockout cell line. Wild-type HeLa and NR4A2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab327018 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Nurr1 antibody [EPR26338-73] (<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 30 µg

Lane 2:

NR4A2 knock-out HeLa lysate at 30 µg

Predicted band size: 67 kDa

false

• WB

Lab

Western blot - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

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

This antibody does not cross-react with overexpressed human NR4A1 or NR4A3 by western blot.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (45-67KDa); Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Nurr1 antibody [EPR26338-73] (<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 10 µg

Lane 2:

293T cells transfected with a human Nurr1 expression vector containing a myc-His-tag® whole cell lysate at 10 µg

Lane 3:

293T cells transfected with a human NR4A3 expression vector containing a myc-His-tag® whole cell lysate at 10 µg

Lane 4:

293T cells transfected with a human NR4A1 expression vector containing a myc-His-tag® whole cell lysate at 10 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 1/100000 dilution

Observed band size: 45-67 kDa,36 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

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

Low expression : liver, colon.

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Nurr1 antibody [EPR26338-73] (<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a>) at 1/1000 dilution

Lane 1:

Human adrenal gland lysate at 20 µg

Lane 2:

Human liver lysate at 20 µg

Lane 3:

Human colon 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: 60 kDa,67 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

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

Performed under reducing conditions.

In Western blot, ab327018 was shown to bind specifically to Nurr1. Target of interest was observed at 60 kDa and 67 KDa in induced wild-type HeLa cell lysates (lane 2) with no signal observed at these sizes in Nurr1 knockout cell line (lanes 3-4).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Nurr1 antibody [EPR26338-73] (<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) was starved for 2 hours, whole cell lysate at 20 µg

Lane 2:

Wild-type HeLa was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate at 20 µg

Lane 3:

Nurr1 knockout HeLa was starved for 2 hours, whole cell lysate at 20 µg

Lane 4:

Nurr1 knockout HeLa was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell 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

Observed band size: 60 kDa,67 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Nurr1 antibody [EPR26338-73] (<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) was starved for 2 hours, whole cell lysate at 80 µg

Lane 2:

NIH/3T3 was starved for 2 hours, and then treated with 200 nM PMA for 2 hours, whole cell lysate at 80 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

Observed band size: 60 kDa,67 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

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

Low expression : pancreas, liver, brain.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Nurr1 antibody [EPR26338-73] (<a href='/en-us/products/primary-antibodies/nurr1-antibody-epr26338-73-ab327018'>ab327018</a>) at 1/1000 dilution

Lane 1:

Mouse pancreas tissue lysate at 80 µg

Lane 2:

Mouse liver tissue lysate at 80 µg

Lane 3:

Mouse adrenal gland tissue lysate at 80 µg

Lane 4:

Mouse brain tissue lysate at 80 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

Observed band size: 60 kDa,67 kDa,36 kDa

false

Exposure time: 180s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling Nurr1 with ab327018 at 1/1000 (0.501 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse cardiac muscle (PMID : 1491694).

The primary antibody was incubated for 30 mins at room temperature. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (fresh frozen) tissue labeling Nurr1 with ab327018 at 1/50 (10.02 μg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 μg/mL dilution (Green).

Negative control : confocal image showing no staining on mouse liver (PMID : 1491694). The nuclear counterstain was DAPI (Blue). The section was incubated with ab327018 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nurr1 antibody [EPR26338-73] - BSA and Azide free (AB326956)

This data was developed using ab327018, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Nurr1 with ab327018 at 1/1000 (0.501 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse liver (PMID : 1491694).

The primary antibody was incubated for 30 mins at room temperature. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

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