PRCP Antibody Protocol: WB, IHC & IF Applications

PRCP Antibody is a research-grade immunological tool designed for the specific detection of prolylcarboxypeptidase (PRCP) protein across multiple applications including Western blot, immunohistochemistry, and immunofluorescence.

Product Name

PRCP Antibody

Catalog Number

Ab349917

Target Protein

Prolylcarboxypeptidase (PRCP)

Recommended Applications

Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)

Supplier Reference

absea#OC-621

Species Reactivity

Please verify with product datasheet; flag for review

Materials and Reagents

Before beginning the protocol, ensure the following materials are available:

• PRCP Antibody (Ab349917, Aladdin Scientific; supplier ref: absea#OC-621)
• Appropriate secondary antibody conjugated to HRP (for WB/IHC) or fluorophore (for IF)
• PBS or TBS buffer (pH 7.4)
• Blocking buffer: 5% non-fat dry milk or 5% BSA in TBST for WB; normal serum for IHC/IF
• Antigen retrieval buffer (for IHC; e.g., citrate buffer pH 6.0 or EDTA buffer pH 9.0)
• Mounting medium appropriate to application
• Standard SDS-PAGE and transfer equipment (for WB)
• Appropriate tissue sections or cultured cells fixed on slides (for IHC/IF)

Note: Refer to the official product datasheet for confirmed species reactivity, recommended dilutions, and storage conditions prior to use.

Step-by-Step Protocol

Western Blot (WB)

1. Prepare cell or tissue lysates using standard RIPA lysis buffer supplemented with protease inhibitors.
2. Determine protein concentration (e.g., BCA assay) and load 20–40 µg total protein per lane on SDS-PAGE gel.
3. Transfer separated proteins to PVDF or nitrocellulose membrane.
4. Block membrane for 1 hour at room temperature with blocking buffer.
5. Incubate with PRCP Antibody at the recommended dilution (refer to product datasheet) overnight at 4°C with gentle agitation.
6. Wash membrane 3 × 10 minutes with TBST.
7. Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.
8. Wash 3 × 10 minutes with TBST, then detect signal using ECL chemiluminescence reagent.

Immunohistochemistry (IHC)

1. Deparaffinize and rehydrate FFPE sections through graded alcohols to water.
2. Perform antigen retrieval using appropriate buffer (heat-induced method recommended).
3. Block endogenous peroxidase activity with 3% H₂O₂ for 10 minutes.
4. Block non-specific binding with normal serum for 30 minutes.
5. Apply PRCP Antibody at recommended dilution; incubate overnight at 4°C in a humidified chamber.
6. Apply secondary antibody and detection reagent per kit instructions.
7. Counterstain with hematoxylin, dehydrate, and mount.

Immunofluorescence (IF)

1. Fix cells on coverslips with 4% paraformaldehyde for 15 minutes, then wash with PBS.
2. Permeabilize with 0.1–0.3% Triton X-100 in PBS for 10 minutes (if detecting intracellular target).
3. Block with 5% normal serum in PBS for 30 minutes at room temperature.
4. Incubate with PRCP Antibody at recommended dilution for 1 hour at room temperature or overnight at 4°C.
5. Wash 3 × 5 minutes with PBS.
6. Apply fluorophore-conjugated secondary antibody for 1 hour at room temperature, protected from light.
7. Counterstain nuclei with DAPI if desired, mount, and image by fluorescence microscopy.

Troubleshooting

The following table summarizes common issues encountered when using the PRCP Antibody and recommended solutions:

• No signal detected: Verify antibody dilution and incubation conditions. Confirm target protein is expressed in your cell/tissue model. Check secondary antibody compatibility and detection system functionality.
• High background: Increase blocking time or blocking agent concentration. Optimize antibody dilution. Increase wash stringency (number of washes or TBST detergent concentration).
• Non-specific bands (WB): Titrate primary antibody concentration. Ensure complete blocking. Consider switching blocking agent (milk vs. BSA).
• Weak signal (IHC/IF): Optimize antigen retrieval method and duration. Increase primary antibody concentration or extend incubation time.
• High autofluorescence (IF): Use autofluorescence quenching reagents. Ensure tissue sections are fresh and properly fixed.

For application-specific dilution ranges and positive control suggestions, always consult the official PRCP Antibody (Ab349917) product datasheet.

Data Interpretation and Expected Results

PRCP (Prolylcarboxypeptidase), also known as angiotensinase C, is a lysosomal serine protease involved in the cleavage of C-terminal amino acids from peptides with a penultimate proline residue. It plays roles in blood pressure regulation, inflammation, and metabolism.

Western Blot: The PRCP protein is expected to appear at its predicted molecular weight (approximately 55–58 kDa for the mature form; verify exact size on product datasheet). Ensure size matches predicted molecular weight accounting for possible glycosylation.

IHC: Positive staining is expected in tissues with documented PRCP expression (e.g., kidney, lung, endothelial cells). Staining pattern should be predominantly cytoplasmic/lysosomal.

IF: Punctate cytoplasmic staining consistent with lysosomal localization may be observed. Co-staining with lysosomal markers can confirm specificity.

Disclaimer: Expected molecular weight and expression pattern are based on published literature and databases. Always validate against appropriate positive and negative controls in your experimental system.

Frequently asked questions

What applications is the PRCP Antibody (Ab349917) validated for?

The PRCP Antibody (Ab349917) is recommended for use in Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) applications. Please consult the official product datasheet for confirmed validated applications and recommended dilutions.

What is the recommended storage condition for PRCP Antibody?

Storage conditions should be confirmed on the official product datasheet for Ab349917. Generally, antibodies are stored at -20°C for long-term storage, with short-term storage at 4°C. Avoid repeated freeze-thaw cycles to maintain activity.

Which species does the PRCP Antibody react with?

Species reactivity information for Ab349917 should be verified on the official product datasheet. Please contact Aladdin Scientific technical support or refer to the datasheet for confirmed reactivity against human, mouse, rat, or other species.

How should I optimize the primary antibody dilution for my application?

Start with the dilution range recommended on the product datasheet as a reference. Perform a titration experiment using serial dilutions to identify the optimal concentration that gives maximum specific signal with minimal background in your specific experimental system.

Can PRCP Antibody be used for co-immunoprecipitation (Co-IP)?

Co-IP compatibility for Ab349917 is not confirmed in the available source material. Please consult the official product datasheet or contact Aladdin Scientific technical support to verify if Co-IP has been validated for this antibody.

References

1. Odya CE, Erdös EG. Human prolylcarboxypeptidase. Methods Enzymol. 1981;80:460–466.
2. Shariat-Madar Z, Mahdi F, Schmaier AH. Identification and characterization of prolylcarboxypeptidase as an endothelial cell prekallikrein activator. J Biol Chem. 2002;277(20):17962–17969.
3. Aladdin Scientific. PRCP Antibody Product Datasheet, Cat. No. Ab349917. Accessed 2024.

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