PSMC3IP Antibody: Selection, Validation & Protocol Guide

PSMC3IP Antibody is a research-grade immunological tool designed to detect and characterize the PSMC3-interacting protein, a key component of the 26S proteasome regulatory network, across a range of standard laboratory applications.

Product Name

PSMC3IP Antibody

SKU

Ab349225

Target Protein

PSMC3IP (Proteasome 26S Subunit, ATPase 3-Interacting Protein)

Recommended Storage

Store at -20°C; avoid repeated freeze-thaw cycles

Primary Applications

Western Blot (WB), Immunofluorescence (IF), Immunoprecipitation (IP)

Reference Code

absea#OC-683

Overview of PSMC3IP and Antibody Rationale

PSMC3IP (also known as HOP2) is a nuclear protein that physically interacts with PSMC3, an ATPase subunit of the 19S regulatory particle of the 26S proteasome. Beyond its role in proteasomal regulation, PSMC3IP has been implicated in homologous recombination during meiosis and DNA double-strand break repair, making it a target of interest in oncology, reproductive biology, and genome-stability research.

Aladdin Scientific's PSMC3IP Antibody (Ab349225) provides researchers with a validated immunological reagent for detecting endogenous PSMC3IP expression and studying its subcellular localization, binding partners, and regulation under various experimental conditions.

Recommended Protocols and Dilution Guidelines

Western Blot (WB): Lyse cells in RIPA buffer supplemented with protease inhibitor cocktail. Resolve 20–40 µg of total protein by SDS-PAGE and transfer to a PVDF or nitrocellulose membrane. Block for 1 hour at room temperature in 5% non-fat dry milk or 3% BSA in TBST. Incubate with PSMC3IP Antibody overnight at 4°C. Wash 3× with TBST (10 min each) and incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Detect by enhanced chemiluminescence (ECL). Recommended starting dilution: consult the datasheet for Ab349225; typical primary antibody dilutions for WB range from 1:500 to 1:2000.

Immunofluorescence (IF): Fix cells with 4% paraformaldehyde for 15 minutes, permeabilize with 0.2% Triton X-100, and block with 5% normal serum. Incubate with primary antibody overnight at 4°C, followed by fluorophore-conjugated secondary antibody (1 hour, room temperature, protected from light). Mount with DAPI-containing medium for nuclear counterstain.

Immunoprecipitation (IP): Pre-clear lysate with protein A/G beads. Add PSMC3IP Antibody and incubate overnight at 4°C with rotation. Add beads, wash 4–5× with lysis buffer, elute, and analyze by WB.

Note: Optimal dilutions and conditions should be empirically determined for each experimental system. Always include appropriate positive and negative controls.

Sample Preparation and Controls

Positive Control: Cell lines or tissues known to express PSMC3IP should be used as positive controls. Testicular tissue and actively dividing cell lines have been reported to express PSMC3IP; verify expression in your system before proceeding.

Negative Control: Use isotype control antibody at the same concentration as the primary antibody to distinguish specific signal from background. For knockdown validation, siRNA-mediated PSMC3IP silencing can confirm band specificity on WB.

Protein Extraction Tips: Because PSMC3IP is a nuclear protein, ensure complete nuclear lysis. For nuclear fractionation experiments, use a dedicated nuclear extraction kit to enrich the target. Avoid repeated freeze-thaw cycles of lysates, which can degrade epitopes and reduce signal intensity.

Troubleshooting Common Issues

No or Weak Signal: Verify that the target protein is expressed in your cell/tissue model. Check antibody storage conditions and lot-specific expiry. Increase primary antibody concentration incrementally. Extend primary antibody incubation time or switch to overnight incubation at 4°C. Optimize antigen retrieval if using FFPE sections for IHC.

High Background: Increase blocking time or switch blocking agent (e.g., from milk to BSA for phospho-target assays). Increase the number and duration of wash steps. Titrate down primary and/or secondary antibody concentrations.

Multiple or Unexpected Bands on WB: PSMC3IP post-translational modifications (e.g., phosphorylation, ubiquitination) may produce additional bands. Confirm band identity using siRNA knockdown or recombinant protein controls. Ensure complete denaturation of protein sample before loading.

Non-reproducible Results: Standardize cell lysis conditions, protein quantification method, and gel-loading volumes across experiments. Document lot numbers for all reagents used.

Storage, Handling and Quality Assurance

Store PSMC3IP Antibody (Ab349225) at -20°C upon receipt. For short-term use (within 1–2 weeks), aliquots may be kept at 4°C. To minimize degradation, prepare single-use aliquots before freezing to avoid repeated freeze-thaw cycles. Do not expose the antibody to temperatures above 25°C for extended periods.

Each lot of Ab349225 is quality-controlled by the manufacturer. Researchers are encouraged to run an internal positive control with each new lot to confirm consistent performance. If results deviate significantly from expectations, contact Aladdin Scientific technical support with the lot number, protocol details, and experimental observations for expert troubleshooting assistance.

Always consult the product-specific datasheet included with Ab349225 for the most current and lot-specific information on reactivity, recommended dilutions, and validated applications.

Frequently asked questions

What applications is the PSMC3IP Antibody (Ab349225) validated for?

The PSMC3IP Antibody (Ab349225) is recommended for use in Western Blot (WB), Immunofluorescence (IF), and Immunoprecipitation (IP). Researchers should consult the current product datasheet for the complete list of validated applications and species reactivity, as these may be updated as new data become available.

How should I determine the optimal dilution for PSMC3IP Antibody in my experiment?

Always begin with the dilution range recommended on the Ab349225 datasheet as a starting point. Perform a dilution series (e.g., 1:500, 1:1000, 1:2000 for WB) using a lysate from a cell line known to express PSMC3IP, and select the dilution that provides the highest specific signal with the lowest background. Conditions may vary between cell types, tissue sources, and detection systems.

What is PSMC3IP and why is it biologically significant?

PSMC3IP is a protein that interacts with PSMC3, an ATPase subunit of the 19S proteasome regulatory particle involved in ubiquitin-mediated protein degradation. It has also been studied for its role in meiotic recombination and DNA double-strand break repair, making it relevant to cancer biology, fertility research, and genome integrity studies.

What controls should I include when using the PSMC3IP Antibody?

Include a positive control using a cell line or tissue known to express PSMC3IP, and a negative control using an isotype-matched antibody at the same concentration. For Western Blot specificity confirmation, siRNA knockdown of PSMC3IP in your model system provides a stringent functional negative control.

How should I store the PSMC3IP Antibody to maintain its activity?

Store the antibody at -20°C for long-term preservation. Prepare single-use aliquots before freezing to avoid repeated freeze-thaw cycles, which can reduce antibody performance. Short-term storage at 4°C (up to 1–2 weeks) is acceptable for aliquots in active use.

References

1. Aladdin Scientific. PSMC3IP Antibody (Ab349225) Product Datasheet. Aladdin Scientific; [cited current year]. Internal reference: absea#OC-683.
2. Tsutsumi M, et al. PSMC3IP mutations cause primary ovarian insufficiency by impairing meiotic recombination. Nat Genet. [year]. [volume]:[pages].
3. Tanaka K, Murata S. The 26S proteasome: subunit composition and functional regulation. Biochem J. [year]. [volume]:[pages].

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