Anti-Glucocorticoid Receptor antibody [EPR19621]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Glucocorticoid Receptor with ab183127 at 1/500 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Flow cytometry overlay histogram showing wild-type (WT) HeLa (green line) and NR3C1 knockout (KO) HeLa stained with ab183127 (magenta line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 mins. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody ab183127 (1x 10⁶ in 100μl at 0.04 μg/ml (1/54,850 dilution) for 30 mins at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30 mins at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (HeLa WT - black line, NR3C1 KO HeLa - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in HeLa WT Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 mins under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling Glucocorticoid Receptor with ab183127 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on tumor cells of the cervix carcinoma is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Immunohistochemical analysis of paraffin-embedded Human glioma tissue labeling Glucocorticoid Receptor with ab183127 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on tumor cells of the Human glioma is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Glucocorticoid Receptor with ab183127 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The results show signal translocation after dexamethasone (100 nM for 2 hours) treatment on Hela cells. PMID : 24291004.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab183127 at 1/500 dilution followed by ab150120 at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Immunohistochemical analysis of paraffin-embedded Rat hippocampus tissue labeling Glucocorticoid Receptor with ab183127 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on rat hippocampus is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Glucocorticoid Receptor with ab183127 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on hepatocytes of the mouse liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Lanes 1-4 : Merged signal (red and green). Green - ab183127 observed at 90-100 kDa. Red - loading control ab8245 observed at 37 kDa.

ab183127 Anti-Glucocorticoid Receptor antibody [EPR19621] was shown to specifically react with Glucocorticoid Receptor in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261766 (knockout cell lysate ab257009) was used. Wild-type and Glucocorticoid Receptor knockout samples were subjected to SDS-PAGE. ab183127 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) 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-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

NR3C1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human NR3C1 (Glucocorticoid Receptor) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-nr3c1-glucocorticoid-receptor-knockout-hela-cell-line-ab261766'>ab261766</a>)

Lane 3:

Wild-type A549 cell lysate at 20 µg

Lane 4:

NR3C1 knockout A549 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 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 90-100 kDa

false

• WB

Supplier Data

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Blocking/Dilution buffer : 5% NFDM/TBST.

This antibody may recognize eight isoforms. The predicted MW are from 61KDa to 86KDa in human, respectively.

All lanes:

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/2000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

A431 (Human epidermoid carcinoma cell line) 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 1/100000 dilution

Predicted band size: 85 kDa

Observed band size: 83 kDa,86 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1 : 30 seconds; Lane 2 : 15 seconds.

This antibody may recognize eight isoforms. The predicted MW are from 61KDa to 86KDa in human, respectively.

All lanes:

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/2000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 85 kDa

Observed band size: 83 kDa,86 kDa

false

• WB

Lab

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Lanes 1 - 4 : Merged signal (red and green). Green - ab183127 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab183127 was shown to recognize NR3C1 in wild-type A549 cells as signal was lost at the expected MW in NR3C1 knockout cell line ab261862 (knockout cell lysate ab261671). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and NR3C1 knockout samples were subjected to SDS-PAGE. ab183127 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/1000 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.

All lanes:

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/2000 dilution

Lane 1:

Wild-type A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

NR3C1 knockout A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human NR3C1 (Glucocorticoid Receptor) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-nr3c1-glucocorticoid-receptor-knockout-a549-cell-line-ab261862'>ab261862</a>)

Lane 3:

A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

Predicted band size: 85 kDa

false

• WB

Supplier Data

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (AB183127)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 0.5 second.

All lanes:

Western blot - Anti-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/20000 dilution

Lane 1:

Empty vector with GFP-Myc tag (vector control) transfected HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate transfected with human Glucocorticoid Receptor with GFP-Myc tag at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 85 kDa

Observed band size: 112 kDa

false