All lanes : Anti-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NR3C1 knockout HeLa cell lysate
Lane 3 : Wild-type A549 cell lysate
Lane 4 : NR3C1 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 86 kDa
Observed band size: 90-100 kDa why is the actual band size different from the predicted?

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

Lane 1 : Wild-type A549 whole cell lysate
Lane 2 : NR3C1 knockout A549 whole cell lysate
Lane 3 : A431 whole cell lysate
Lane 4 : U-87 MG whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 86 kDa

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 cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. 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 : Anti-Glucocorticoid Receptor antibody [EPR19621] (ab183127) at 1/2000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 86 kDa
Observed band size: 83,86 kDa why is the actual band size different from the predicted?

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

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : A431 (Human epidermoid carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 86 kDa
Observed band size: 83,86 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

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 : 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
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate transfected with human Glucocorticoid Receptor with GFP-Myc tag

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 86 kDa
Observed band size: 112 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 0.5 second.

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.

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.

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.

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.

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.

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.