MA5-16394

antibody from Invitrogen Antibodies

Targeting: PGR NR3C3, PR

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Flow cytometry (Recommended by provider)

Antibody Data

Product number

MA5-16394

Provider

Invitrogen Antibodies

Product name

Progesterone Receptor Monoclonal Antibody (SP42)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 30 min at room temperature. A suggested positive control is breast carcinoma.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

SP42

Vial size

500 µL

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockout of Progesterone Receptor was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1031637_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Progesterone Receptor was performed by loading 30 µg of T-47D wild type (Lane 1), T-47D Cas9 (Lane 2) andT-47D Progesterone Receptor KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Progesterone Receptor Monoclonal Antibody (SP42) (Product # MA5-16394, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:8000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Progesterone Receptor.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Progesterone Receptor Monoclonal Antibody (Product # MA5-16394) and a ~110 kDa and ~82 kDa band corresponding to Progesterone Receptor was observed in T-47D and uterus tissue. Additional un-characterized bands (*) were also observed. Whole cell extracts (30ug lysate) of T-47D (Lane 1), SK-BR-3 (Lane 2), Mouse Lung (Lane 3), Rat Lung (Lane 4), Mouse Uterus (Lane 5) and Rat Uterus (Lane 6) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Progesterone Receptor was performed using 70% confluent log phase T-47D cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Progesterone Receptor Monoclonal Antibody (Product # MA5-16394) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). 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. Panel e represents SK-BR-3 cells having no expression of Progesterone Receptor. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of Progesterone Receptor using anti-Progesterone Receptor Monoclonal Antibody (Product # MA5-16394) in Breast Carcinoma Cancer Tissue. The recommened dilution for this antibody in immunohistochemistry applications is 1:400.