Anti-Glucocorticoid receptor antibody [EPR24889-231]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid receptor antibody [EPR24889-231] (AB305050)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling Glucocorticoid receptor with ab305050 at 1/100 (5.12 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on human pancreas (PMID : 32619553). The section was incubated with ab305050 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid receptor antibody [EPR24889-231] (AB305050)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Glucocorticoid receptor with ab305050 at 1/100 (5.12 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on human kidney. The section was incubated with ab305050 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid receptor antibody [EPR24889-231] (AB305050)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Glucocorticoid receptor with ab305050 at 1/100 (5.12 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on human liver. The section was incubated with ab305050 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

• ICC

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Immunocytochemistry - Anti-Glucocorticoid receptor antibody [EPR24889-231] (AB305050)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NR3C1 KO HeLa (NR3C1 knockout human cervical adenocarcinoma epithelial cell) (ab261766) cells labelling Glucocorticoid receptor with ab305050 at 1/50 (10.24 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 no staining in NR3C1 knockout HeLa cells and showing nuclear and weak cytoplasmic staining in wildtype HeLa cells. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Red). 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.

• IP

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Immunoprecipitation - Anti-Glucocorticoid receptor antibody [EPR24889-231] (AB305050)

Glucocorticoid receptor was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab305050 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305050 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2 : ab305050 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab305050 in HeLa whole cell lysate

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

Exposure time : 6 seconds

The bands beneath the target band (94 kDa) are likely to be degraded target fragments

All lanes:

Immunoprecipitation - Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2:

ab305050 IP in HeLa 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

Predicted band size: 94 kDa

Observed band size: 94 kDa

false

Exposure time: 6s

• WB

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Western blot - Anti-Glucocorticoid receptor antibody [EPR24889-231] (AB305050)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The bands beneath the target band (94 kDa) in lane 3 are likely to be degraded target fragments. Lysates were freshly made and used immediately to minimize protein degradation. Exposure time : Lane 1 : 37 seconds Lanes 2-3 : 3 minutes

All lanes:

Western blot - Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lane 2:

293T (human embryonic kidney epithelial cell), whole cell lysate

Lane 3:

THP-1 (human monocytic leukemia monocyte), whole cell lysate

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: 94 kDa

false

• WB

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Western blot - Anti-Glucocorticoid receptor antibody [EPR24889-231] (AB305050)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS The bands beneath the target band (94 kDa) are likely to be degraded target fragments. False colour image of Western blot : Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab305050 was shown to bind specifically to Glucocorticoid receptor. A band was observed at 94 kDa in wild-type HeLa cell lysates with no signal observed at this size in Glucocorticoid receptor knockout cell line. To generate this image, wild-type and Glucocorticoid receptor knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/10000 dilution.

All lanes:

Western blot - Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lane 2:

Glucocorticoid receptor knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lane 3:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate

Secondary

Lanes 1 - 3:

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

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Observed band size: 94 kDa

false