PSMD7 Mouse mAb Protocol: WB, IHC & IP Applications

PSMD7 Mouse mAb is a highly specific monoclonal antibody targeting the PSMD7 subunit of the 26S proteasome regulatory particle, designed for use in Western blotting, immunohistochemistry, and immunoprecipitation to support research into ubiquitin-proteasome pathway function.

Product Name

PSMD7 Mouse mAb

SKU

Ab349226

Host Species

Mouse

Antibody Type

Monoclonal Antibody (mAb)

Target Protein

PSMD7 (26S Proteasome Regulatory Subunit 7)

Recommended Applications

Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP)

Reference Number

absea#KC-36422

Background: PSMD7 and the 26S Proteasome

PSMD7 (also known as MOV34 or S12) is a non-ATPase regulatory subunit of the 19S regulatory particle (RP) of the 26S proteasome. The 26S proteasome is the principal ATP-dependent protease responsible for the selective degradation of ubiquitinated proteins in eukaryotic cells, playing essential roles in protein quality control, cell cycle progression, DNA damage response, and signal transduction.

PSMD7 contains a conserved MPN (Mpr1-Pad1-N-terminal) domain, which is implicated in protein–protein interactions within the proteasome lid complex. Dysregulation of PSMD7 and proteasomal activity has been linked to cancer, neurodegeneration, and inflammatory diseases, making it a relevant target for both basic research and drug discovery programs.

Sample Preparation

Cell Lysate Preparation:

1. Wash cells twice with ice-cold phosphate-buffered saline (PBS).
2. Lyse cells in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail and phosphatase inhibitors.
3. Incubate on ice for 30 minutes with occasional vortexing.
4. Centrifuge at 12,000 × g for 15 minutes at 4°C. Collect the supernatant.
5. Determine protein concentration by BCA or Bradford assay.

Tissue Lysate Preparation:

1. Homogenize fresh or snap-frozen tissue in RIPA buffer containing protease inhibitors using a mechanical homogenizer.
2. Centrifuge at 14,000 × g for 20 minutes at 4°C. Collect the cleared supernatant.
3. Quantify protein and store aliquots at −80°C until use.

Western Blot (WB) Protocol

Gel Electrophoresis and Transfer:

1. Load 20–40 µg of total protein per lane on an appropriate percentage SDS-PAGE gel (recommended: 10–12%).
2. Run electrophoresis and transfer proteins to a PVDF or nitrocellulose membrane.
3. Block the membrane in 5% non-fat dry milk or 5% BSA in TBST (0.1% Tween-20) for 1 hour at room temperature.

Antibody Incubation:

1. Dilute PSMD7 Mouse mAb (Ab349226) in blocking buffer at the manufacturer's recommended dilution (refer to product datasheet; specific dilution to be confirmed).
2. Incubate membrane with primary antibody overnight at 4°C with gentle rocking.
3. Wash three times with TBST, 10 minutes each.
4. Incubate with appropriate HRP-conjugated anti-mouse secondary antibody for 1 hour at room temperature.
5. Wash three times with TBST, 10 minutes each.

Detection:

1. Develop with ECL substrate. Expose to X-ray film or image with a chemiluminescence imager.
2. Expected band size for PSMD7: approximately 37 kDa (confirm with product datasheet).

Immunohistochemistry (IHC) Protocol

Tissue Section Preparation:

1. Fix tissues in 10% neutral buffered formalin and embed in paraffin (FFPE) or use frozen sections as appropriate.
2. Cut sections at 4–5 µm thickness and mount on positively charged slides.
3. For FFPE sections: deparaffinize in xylene and rehydrate through graded ethanol series to water.

Antigen Retrieval and Blocking:

1. Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) for 20 minutes.
2. Cool slides, wash with PBS, and block endogenous peroxidase with 3% H₂O₂ for 10 minutes.
3. Block with 5–10% normal serum (species matched to secondary antibody) for 30 minutes at room temperature.

Antibody Incubation and Detection:

1. Apply PSMD7 Mouse mAb (Ab349226) at the recommended dilution (refer to product datasheet). Incubate overnight at 4°C in a humidified chamber.
2. Wash with PBS three times, then apply appropriate secondary antibody with polymer-HRP detection system.
3. Develop with DAB chromogen, counterstain with hematoxylin, dehydrate, and mount coverslip.

Immunoprecipitation (IP) Protocol and Troubleshooting

Immunoprecipitation Protocol:

1. Pre-clear 500 µg–1 mg of cell lysate with Protein A/G agarose beads for 1 hour at 4°C with rotation. Collect pre-cleared supernatant.
2. Add PSMD7 Mouse mAb (Ab349226) at the recommended amount (refer to product datasheet; typically 1–5 µg per IP reaction). Incubate at 4°C overnight with rotation.
3. Add Protein A/G beads and incubate for an additional 2 hours at 4°C with rotation.
4. Centrifuge at 1,000 × g for 5 minutes. Discard supernatant and wash beads four times with ice-cold lysis buffer.
5. Elute by boiling beads in 2× SDS loading buffer for 5 minutes. Resolve by SDS-PAGE and analyze by Western blot.

Troubleshooting Guide:

Problem	Possible Cause	Solution
No signal on WB	Insufficient antigen; over-blocking	Increase protein loading; reduce blocking time or concentration
High background	Insufficient washing; excess antibody	Increase wash steps; optimize antibody dilution
Non-specific bands	Cross-reactivity; degradation	Use fresh lysate with protease inhibitors; titrate antibody
Weak IHC signal	Inadequate antigen retrieval	Optimize retrieval buffer pH and duration

Frequently asked questions

What applications is PSMD7 Mouse mAb (Ab349226) validated for?

PSMD7 Mouse mAb (Ab349226) is recommended for use in Western blotting (WB), immunohistochemistry (IHC), and immunoprecipitation (IP). Please refer to the official product datasheet for confirmed validation data and species reactivity.

What is the expected molecular weight of PSMD7 detected by this antibody?

PSMD7 has a theoretical molecular weight of approximately 37 kDa. The observed band on Western blot may vary slightly depending on the cell or tissue type and post-translational modifications. Always confirm with the product datasheet.

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

Heat-induced epitope retrieval (HIER) is generally recommended. Citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) are common starting points. The optimal retrieval conditions should be determined empirically and confirmed against the product datasheet.

What controls should be included when using PSMD7 Mouse mAb?

Include a positive control lysate or tissue known to express PSMD7, an isotype control (mouse IgG at the same concentration) to assess non-specific binding, and a negative control with no primary antibody to monitor secondary antibody background.

How should PSMD7 Mouse mAb (Ab349226) be stored?

Monoclonal antibodies are generally stored at −20°C for long-term preservation and at 4°C for short-term use. Avoid repeated freeze-thaw cycles. Refer to the product datasheet for specific storage conditions and expiry information for Ab349226.

References

1. Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem. 2009;78:477-513. doi:10.1146/annurev.biochem.78.081507.101607
2. Bhatt NY, et al. The 26S proteasome: mechanisms of substrate selection and degradation. Curr Opin Struct Biol. General reference — specific citation details require verification.
3. Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002;82(2):373-428. doi:10.1152/physrev.00027.2001

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