Recombinant 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.

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

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Glucocorticoid receptor with ab305050 at 1/100 dilution (5.12 µg/ml), followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection). Nuclear staining on human liver is observed. 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 Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins. 

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling Glucocorticoid receptor with ab305050 at 1/100 dilution (5.12 µg/ml), followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection). Nuclear staining on human pancreas is observed (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 Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Glucocorticoid receptor with ab305050 at 1/100 dilution (5.12 µg/ml), followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection). Nuclear staining on human kidney is observed. 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 Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins. 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NR3C1 KO HeLa (NR3C1 knockout human cervical adenocarcinoma epithelial cell) (ab261766) cells labeling Glucocorticoid receptor with AB305050 at 1/50 dilution (10.24 µg/mL), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/mL) (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). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/mL) (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 dilution (2 µg/mL).

Glucocorticoid receptor was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab305050 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305050 at 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 µg (Inset)

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

Observed M_W (kDa): 94. 

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

Chromatin was prepared from A549+dexamethasone(100 nM 1h) cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
The ChIP was performed with 25 µg of chromatin, 5 µg of 305050 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads.

The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol