Anti-Glucocorticoid Receptor antibody [EPR19621] - BSA and Azide free

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid Receptor antibody [EPR19621] - BSA and Azide free (AB223138)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183127).

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] - BSA and Azide free (AB223138)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183127).

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] - BSA and Azide free (AB223138)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183127).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Glucocorticoid Receptor antibody [EPR19621] - BSA and Azide free (AB223138)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183127).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Glucocorticoid Receptor antibody [EPR19621] - BSA and Azide free (AB223138)

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid Receptor antibody [EPR19621] - BSA and Azide free (AB223138)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183127).

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] - BSA and Azide free (AB223138)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183127).

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] - BSA and Azide free (AB223138)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/glucocorticoid-receptor-antibody-epr19621-ab183127'>ab183127</a>) 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

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