PA1-512

antibody from Invitrogen Antibodies

Targeting: NR3C1 GR, GRL

Provider product page for PA1-512

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Gel shift

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [42]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [3]
Other assay [2]

Submit

Product number: PA1-512 - Provider product page
Provider: Invitrogen Antibodies
Product name: Glucocorticoid Receptor Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-512 detects glucocorticoid receptor (GR) from human, mouse and rat tissues. PA1-512 has been successfully used in Western blot, immunohistochemistry, immunocytochemistry, immunoprecipitation, immunofluorescence, ChIP and gel shift procedures. By Western blot, this antibody detects an ~97 kDa protein representing GR as well as two other unidentified proteins at ~135 to ~145 kDa from rat liver extract. These cross reacting proteins are not believed to be GR or GR precursors as they do not bind [3H]dexamethasone mesylate. Immunohistochemical staining of GR in rat hippocampus using PA1-512 results in staining primarily in the nucleus. PA1-512 does not disrupt receptor-DNA interactions. The PA1-512 immunogen is a synthetic peptide corresponding to residues C(245) K P L I L P D T K P K I K D(259) of human GR. The PA1-512 immunizing peptide (Cat. # PEP-002) is available for use in for neutralization and control experiments. Reconstitute in 50 µL PBS to create a stock of 1 mg/mL.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 50 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

NR3C1 protein structure - PA1-512 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Glucocorticoid Receptor was performed by loading 20 µg of the indicated whole cell lysates and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. Glucocorticoid Receptor was detected at ~95 kDa using a Glucocorticoid Receptor rabbit polyclonal antibody (Product # PA1-512) at a concentration of 1 µg/mL in blocking buffer overnight at 4C on a rocking platform, followed by a Goat anti-rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a dilution of 1:1000 for at least one hour at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Glucocorticoid Receptor was achieved by transfecting MDA-MB-231 with Glucocorticoid Receptor specific siRNAs (Silencer® select Product # s6186 and s6187). Western blot analysis (Fig. a) was performed using whole cell extracts from the Glucocorticoid Receptor knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Glucocorticoid Receptor Polyclonal Antibody (Product # PA1-512, 0.15 µg/ml) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Glucocorticoid Receptor.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Glucocorticoid Receptor Polyclonal Antibody (Product # PA1-512) and a 86 kDa band corresponding to Glucocorticoid Receptor was observed across cell lines and tissues. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), MDA-MB-231 (Lane 2), MCF7 (Lane 3), DU 145 (Lane 4) in Figure a and Mouse Skeletal Muscle (Lane 1), Mouse Brain (Lane 2), Rat Skeletal Muscle (Lane 3) and Rat Heart (Lane 4) in Figure b, were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.75 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Uncharacterized bands (*) were observed at 38 kDa, 55 kDa and 180 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Glucocorticoid Receptor using Glucocorticoid Receptor Polyclonal Antibody (59) (Product # PA1-512) shows staining in A2058 Cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Glucocorticoid Receptor (Product # PA1-512) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Glucocorticoid Receptor using Glucocorticoid Receptor Polyclonal Antibody (59) (Product # PA1-512) shows staining in Hela Cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Glucocorticoid Receptor (Product # PA1-512) at a dilution of 1:100 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Glucocorticoid Receptor was performed using MDA-MB-231 cells (serum-starved) and MDA-MB-231 cells serum-starved for 24 hours, followed by 1 µM Dexamethasone treatment for 2 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Glucocorticoid Receptor Polyclonal Antibody (Product # PA1-512) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Green) in MDA-MB-231 treated cells. Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization of NR3C1 protein in MDA-MB-231 treated cells. Panel e represents the merged image of MDA-MB-231 untreated cells, that shows cytoplasmic localization of NR3C1 protein. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.