Progesterone Receptor Mouse mAb Protocol | Detection & Usage

Progesterone Receptor Mouse mAb is a high-specificity monoclonal antibody designed for the reliable immunodetection of the progesterone receptor (PR) protein in human and mammalian samples across multiple assay platforms including IHC, WB, and IF.

Product Name

Progesterone Receptor Mouse mAb

SKU / Catalog Number

Ab349219

Host Species

Mouse

Antibody Type

Monoclonal Antibody (mAb)

Target Protein

Progesterone Receptor (PR)

Recommended Applications

IHC, WB, IF (verify with datasheet)

Reference Clone / Internal Code

absea#KC-1153

Overview of Progesterone Receptor and Antibody

The progesterone receptor (PR) is a nuclear hormone receptor encoded by the PGR gene. Upon ligand binding, PR translocates to the nucleus and acts as a transcription factor regulating genes involved in reproductive biology, mammary gland development, and uterine function. Two major isoforms, PR-A and PR-B, arise from alternative promoter usage within the same gene and exhibit distinct transcriptional activities.

Immunodetection of PR is clinically significant in breast and endometrial cancer pathology, where PR status guides hormone therapy decisions. Progesterone Receptor Mouse mAb (Ab349219, internal reference absea#KC-1153) is a monoclonal antibody raised in mouse that provides high specificity for PR, minimizing cross-reactivity with structurally related steroid hormone receptors.

Materials and Equipment Required

• Progesterone Receptor Mouse mAb (Ab349219)
• Appropriate secondary antibody (anti-mouse IgG, HRP- or fluorophore-conjugated)
• Positive control tissue or cell lysate known to express PR (e.g., MCF-7 breast cancer cells)
• Negative control (PR-negative tissue or isotype control antibody)
• PBS (pH 7.4) or TBS wash buffer
• Blocking buffer (e.g., 5% non-fat dry milk in TBST for WB; 3–5% normal serum or BSA in PBS for IHC/IF)
• Antigen retrieval reagent for IHC (e.g., citrate buffer pH 6.0 or EDTA buffer pH 9.0)
• Microwave or pressure cooker / water bath for heat-induced epitope retrieval (HIER)
• SDS-PAGE system and PVDF or nitrocellulose membrane for WB
• Mounting medium with DAPI (for IF)
• DAB chromogen kit (for IHC)

Step-by-Step Protocol

A. Immunohistochemistry (IHC)

1. Tissue preparation: Fix tissue in 10% neutral-buffered formalin and embed in paraffin (FFPE). Section at 3–5 µm and mount on positively charged slides.
2. Deparaffinization and rehydration: Bake sections at 60°C for 30 min, dewax in xylene (3 × 5 min), and rehydrate through graded ethanol to distilled water.
3. Antigen retrieval (HIER): Submerge slides in pre-heated citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) and heat for 10–20 min. Allow to cool for 20 min at room temperature.
4. Endogenous peroxidase quenching: Incubate with 3% H₂O₂ in methanol for 10 min; wash 3 × 5 min in PBS.
5. Blocking: Apply 3–5% normal serum (matched to secondary antibody host) or 1% BSA in PBS for 30–60 min at room temperature.
6. Primary antibody incubation: Apply Progesterone Receptor Mouse mAb (Ab349219) at the manufacturer-recommended dilution (see datasheet). Incubate overnight at 4°C in a humidified chamber.
7. Washing: Wash 3 × 5 min in PBS.
8. Secondary antibody: Apply HRP-conjugated anti-mouse IgG secondary antibody at recommended dilution; incubate 30–60 min at room temperature.
9. Detection: Apply DAB substrate; monitor colour development under microscope (typically 2–10 min); stop with water.
10. Counterstain and mount: Counterstain with haematoxylin, dehydrate, clear in xylene, and coverslip with permanent mounting medium.

B. Western Blot (WB)

1. Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitor cocktail. Determine protein concentration by BCA or Bradford assay. Load 20–50 µg total protein per lane.
2. SDS-PAGE: Separate proteins on an 8–10% SDS-PAGE gel; transfer to PVDF membrane (pre-activated in methanol).
3. Blocking: Block membrane with 5% non-fat milk in TBST for 1 h at room temperature.
4. Primary antibody: Incubate with Progesterone Receptor Mouse mAb (Ab349219) at manufacturer-recommended dilution overnight at 4°C with gentle rocking.
5. Washing and secondary antibody: Wash 3 × 10 min in TBST; incubate with HRP-conjugated anti-mouse IgG for 1 h at room temperature.
6. Detection: Wash 3 × 10 min in TBST; develop with ECL substrate and image. Expected band: ~94 kDa (PR-B) and/or ~81 kDa (PR-A).

C. Immunofluorescence (IF)

1. Cell preparation: Seed cells on coverslips; fix with 4% paraformaldehyde for 15 min at room temperature; permeabilize with 0.1–0.3% Triton X-100 for 10 min.
2. Blocking: Block with 3–5% BSA or normal serum in PBS for 30–60 min.
3. Primary antibody: Apply Ab349219 at recommended dilution; incubate overnight at 4°C.
4. Secondary antibody: Wash 3 × 5 min in PBS; apply fluorophore-conjugated anti-mouse IgG for 1 h at room temperature protected from light.
5. Nuclear counterstain and mounting: Wash, apply DAPI-containing mounting medium, and seal coverslip. Image by fluorescence microscopy.

Troubleshooting

Problem	Possible Cause	Recommended Action
No signal	Inadequate antigen retrieval; antibody too dilute; antigen loss during processing	Optimize HIER conditions (buffer pH, heating time); titrate primary antibody; check tissue fixation time
High background	Insufficient blocking; primary antibody concentration too high; secondary antibody cross-reactivity	Extend blocking step; reduce primary antibody concentration; use more stringent wash conditions
Non-specific bands (WB)	Overloading; incomplete transfer; non-specific secondary binding	Reduce protein load; verify transfer efficiency; use isotype control
Weak nuclear staining (IHC/IF)	Over-fixation; suboptimal antigen retrieval	Reduce fixation time; switch antigen retrieval buffer (e.g., pH 9.0 EDTA)

Storage, Handling and Safety

Store Progesterone Receptor Mouse mAb (Ab349219) according to the conditions specified on the product datasheet. General guidelines for monoclonal antibodies include storage at 4°C for short-term use (up to one month) and at −20°C for long-term storage. Avoid repeated freeze-thaw cycles, as these can denature the antibody and reduce specific activity. Aliquot the antibody upon first thaw to minimise freeze-thaw events.

Handle all biological reagents in accordance with standard laboratory biosafety procedures. Wear appropriate personal protective equipment (PPE) including gloves, lab coat, and eye protection. Consult the Safety Data Sheet (SDS) for disposal guidance. Do not use reagents beyond the stated expiry date.

Frequently asked questions

What positive control should I use for Progesterone Receptor Mouse mAb?

MCF-7 human breast cancer cells or T-47D cells are widely used positive controls for PR immunodetection, as they endogenously express high levels of both PR-A and PR-B isoforms. For tissue sections, breast carcinoma or endometrial tissue with known PR-positive status is recommended.

Which antigen retrieval method is recommended for IHC with this antibody?

Heat-induced epitope retrieval (HIER) is typically recommended for nuclear receptors including PR. Both citrate buffer (pH 6.0) and EDTA/Tris buffer (pH 9.0) can be effective; the optimal conditions should be determined empirically. Please consult the product datasheet for Ab349219 for the manufacturer-validated conditions.

What is the expected molecular weight of progesterone receptor on Western blot?

The progesterone receptor runs at approximately 94 kDa (PR-B isoform) and approximately 81 kDa (PR-A isoform) on SDS-PAGE. Both isoforms may be detected depending on the cell or tissue type used. Note that the apparent molecular weight on gel may differ slightly from the theoretical value due to post-translational modifications.

Can this antibody be used for flow cytometry or ChIP assays?

The suitability of Ab349219 for flow cytometry or chromatin immunoprecipitation (ChIP) has not been confirmed from the source material provided. Please consult the official product datasheet or contact Aladdin Scientific technical support for validated application information before attempting these assays.

How should I store the antibody to maintain optimal activity?

Store Ab349219 at 4°C for short-term use or at −20°C for long-term storage. Aliquot into single-use volumes upon first thaw to avoid repeated freeze-thaw cycles, which degrade antibody performance. Refer to the product datasheet for exact storage buffer composition and shelf-life information.

References

1. Brisken C, O'Malley B. Hormone action in the mammary gland. Cold Spring Harb Perspect Biol. 2010;2(12):a003178. doi:10.1101/cshperspect.a003178
2. Kastner P, Krust A, Turcotte B, et al. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J. 1990;9(5):1603-1614. PMID:2328727
3. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol. 2010;28(16):2784-2795. doi:10.1200/JCO.2009.25.6529

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.