PSMD11 Antibody

What is PSMD11 and why is it significant for research?

PSMD11 (Proteasome 26S subunit, non-ATPase, 11, also known as RPN6, S9, or p44.5) is a non-ATPase regulatory subunit of the 19S/PA700 proteasome lid subcomplex that plays a crucial role in the 26S proteasome complex involved in protein degradation . The significance of PSMD11 in research stems from its essential functions in:

• Regulating the breakdown of ubiquitinated proteins

• Influencing cell cycle control, DNA repair, and immune responses

• Contributing to cancer progression, particularly in lung adenocarcinoma and other malignancies

• Participating in cuproptosis, an emerging form of regulated cell death

• Correlating with immune cell infiltration and immunosuppressive mechanisms in tumors

Research has shown that PSMD11 expression is regulated by FOXO4 in human embryonic stem cells, and increased PSMD11 expression enhances proteasome assembly and activity .

What are the common applications for PSMD11 antibodies in research?

PSMD11 antibodies have been validated for multiple research applications that help scientists investigate its expression, localization, and interactions:

When selecting an application, researchers should consider their experimental goals and the sample type available .

What are the validated reactivity species for PSMD11 antibodies?

Commercial PSMD11 antibodies have been validated for reactivity against multiple species, allowing for comparative studies across model organisms:

• Human: Validated in numerous cell lines (HeLa, MCF-7, 293T, NCI-H460) and tissues

• Mouse: Validated in brain and testis tissues

• Rat: Validated in brain, testis, and lung tissues

This cross-reactivity is based on sequence homology between species. For example, rabbit monoclonal antibodies against PSMD11 are available that have been validated to detect endogenous PSMD11 across human, mouse, rat, and monkey samples .

What are the optimal conditions for using PSMD11 antibodies in Western blotting?

For optimal Western blot results with PSMD11 antibodies, researchers should follow these methodological guidelines:

• Sample Preparation:

  • For cell lines: Lyse cells with Laemmli buffer on ice, collect by scraping, briefly ultrasonicate, and boil for 10 minutes

  • For tissues: Grind in liquid nitrogen, mix with cold protein extraction buffer, ultrasonicate, and boil for 10 minutes

• Gel Electrophoresis and Transfer:

  • Use 10% SDS-PAGE for optimal separation around the 47kDa range

  • Transfer to nitrocellulose membranes

• Blocking and Antibody Incubation:

  • Block membranes with 5% nonfat milk for 1 hour

  • Incubate with primary PSMD11 antibody at recommended dilutions (typically 1:1000 for Cell Signaling Technology antibody or 1:500-1:2000 for Proteintech antibody)

  • Incubate overnight at 4°C

  • Wash thoroughly with TBST buffer

  • Incubate with appropriate secondary antibody for 2 hours at room temperature

• Detection:

  • Use enhanced chemiluminescence substrate for detection

  • Expected molecular weight: 47kDa

  • Analyze band densitometry using software like ImageJ

For recombinant rabbit monoclonal antibodies, manufacturers recommend not aliquoting the antibody to maintain stability and performance .

How should PSMD11 antibodies be used for immunohistochemistry in tissue samples?

For effective immunohistochemical detection of PSMD11 in tissue samples:

• Tissue Preparation:

  • Dewax paraffin sections in xylene

  • Rehydrate in graded concentrations of ethanol and distilled water

  • Block endogenous peroxidase activity with 0.3% H₂O₂ for 10 minutes at room temperature

• Antigen Retrieval:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative: Citrate buffer at pH 6.0

• Blocking and Antibody Application:

  • Block with 10% normal goat serum for 30 minutes

  • Apply PSMD11 antibody at 1:100 dilution (Bioworld Technology) or 1:250-1:1000 dilution (Proteintech)

  • Incubate overnight at 4°C

• Detection and Visualization:

  • Apply appropriate secondary antibody

  • Develop with diaminobenzidine (DAB)

  • Quantitatively score sections based on percentage of positive cells and staining intensity

This protocol has been successfully used to compare PSMD11 expression between tumor and normal tissues in lung adenocarcinoma studies .

What controls should be included when working with PSMD11 antibodies?

To ensure experimental validity and interpretable results, researchers should include these essential controls:

• Positive Controls:

  • Cell lines: HeLa, MCF-7, 293T, or NCI-H460 cells (known to express PSMD11)

  • Tissues: Mouse brain, mouse testis, rat testis, or rat brain tissue

• Negative Controls:

  • Primary antibody omission control

  • Isotype control using rabbit IgG at equivalent concentration

  • Tissues known to have low PSMD11 expression

• Loading Controls:

  • For Western blot: β-actin (1:5000 dilution) or GAPDH (1:5000 dilution)

  • For IHC: Adjacent serial sections with control antibodies

• Knockdown/Overexpression Controls:

  • PSMD11 knockdown cell lines (as used in lung carcinoma PC9 cells)

  • PSMD11 overexpression cell lines (as used in A549 cells)

These controls help validate antibody specificity and rule out non-specific binding or technical artifacts .

How can PSMD11 antibodies be utilized to study its role in cancer progression?

PSMD11 antibodies can be strategically employed to investigate its contributions to cancer biology through several advanced approaches:

What are the methodological considerations for studying PSMD11 in relation to cuproptosis?

Cuproptosis is an emerging form of regulated cell death induced by copper ions that involves mitochondrial metabolism. To investigate PSMD11's role in cuproptosis, researchers should consider:

• Correlation Analysis:

  • Use Western blot to measure PSMD11 expression alongside known cuproptosis markers (DLAT, DLD, PDHA1)

  • Research has established positive correlations between PSMD11 expression and these cuproptosis genes (P<0.001)

• Copper Challenge Experiments:

  • Treat cells with copper ions at varying concentrations

  • Monitor PSMD11 expression changes via Western blot

  • Assess cell viability and markers of cuproptosis

• Mitochondrial Function Assays:

  • Combine PSMD11 antibodies with mitochondrial markers in immunofluorescence

  • Measure mitochondrial membrane potential and metabolism in cells with modulated PSMD11 expression

  • Correlate findings with cuproptosis indicators

• Proteasomal Activity Assessment:

  • Investigate how PSMD11 expression affects proteasome assembly and activity under copper stress

  • Monitor degradation of ubiquitinated proteins during cuproptosis

• In vivo Models:

  • Use IHC to assess PSMD11 expression in tissues from animal models treated with copper

  • Correlate with tissue damage and cuproptosis markers

These methodological approaches can help elucidate the mechanistic relationships between PSMD11, the proteasome system, and cuproptosis pathways in cancer and other diseases .

How should researchers interpret variations in PSMD11 antibody staining patterns?

Interpreting PSMD11 antibody staining patterns requires careful consideration of several factors:

Proper interpretation requires understanding both technical aspects of the staining and the biological context of PSMD11 function .

What are common challenges in PSMD11 antibody applications and how can they be addressed?

Researchers may encounter several challenges when working with PSMD11 antibodies, with specific solutions for each issue:

For recombinant antibodies like the Cell Signaling Technology D1T1R rabbit mAb, manufacturers specifically recommend not aliquoting to maintain optimal performance .

How do different fixation methods affect PSMD11 antibody performance in immunostaining?

Fixation methods significantly impact epitope preservation and accessibility, affecting PSMD11 antibody performance:

• Formalin Fixation:

  • Most common for IHC applications

  • Advantages: Good morphological preservation, compatible with paraffin embedding

  • Limitations: May mask epitopes through protein cross-linking

  • Optimization: Antigen retrieval is crucial; TE buffer pH 9.0 is recommended as primary choice, with citrate buffer pH 6.0 as an alternative

• Methanol/Acetone Fixation:

  • Suitable for immunofluorescence of cultured cells

  • Advantages: Minimal epitope masking, good for cytoplasmic proteins

  • Limitations: Poorer morphological preservation

  • Recommended for detecting PSMD11 in subcellular compartments (cytoplasm, nucleus, cytosol)

• Paraformaldehyde Fixation:

  • Common for immunofluorescence

  • Advantages: Better morphology than methanol/acetone

  • Limitations: May require permeabilization for intracellular targets

  • Optimization: Additional permeabilization with 0.1-0.5% Triton X-100 improves access to PSMD11

• Fresh-Frozen Tissue:

  • Alternative to FFPE for detecting sensitive epitopes

  • Advantages: Minimal epitope alteration

  • Limitations: Poorer morphology, more technically demanding

  • Consider for applications where FFPE processing affects antibody binding

The fixation method should be selected based on the specific experimental goals and subsequent analysis techniques .

What factors should be considered when selecting between polyclonal and monoclonal PSMD11 antibodies?

The choice between polyclonal and monoclonal PSMD11 antibodies significantly impacts experimental outcomes:

Polyclonal PSMD11 Antibodies:

• Examples: Proteintech 14786-1-AP, Assay Genie CAB15306

• Advantages:

  • Recognize multiple epitopes, providing stronger signals

  • More tolerant of minor protein denaturation or modifications

  • Generally more sensitive for Western blot and IHC

• Limitations:

  • Batch-to-batch variability

  • Potential for higher background

  • May show some cross-reactivity

• Best applications: Western blot, IHC of fixed tissues, IP applications

• Typical working dilutions: 1:500-1:2000 (WB), 1:250-1:1000 (IHC)

Monoclonal PSMD11 Antibodies:

• Examples: Cell Signaling Technology D1T1R clone

• Advantages:

  • Consistent lot-to-lot reproducibility

  • Higher specificity for single epitope

  • Cleaner results with less background

  • Superior for quantitative applications

• Limitations:

  • May be more sensitive to epitope masking

  • Potentially weaker signal than polyclonals

• Best applications: Quantitative Western blot, flow cytometry

• Typical working dilutions: 1:1000 (WB)

Recombinant Antibodies:

• The new generation of PSMD11 antibodies (like CST's D1T1R)

• Advantages:

  • Superior lot-to-lot consistency

  • Continuous supply without animal immunization

  • Animal-free manufacturing

• Special considerations: Manufacturer recommends not aliquoting

Selection should be based on the specific application, required specificity, and whether quantitative or qualitative data is needed .

How can PSMD11 antibodies be used to investigate its role in lung adenocarcinoma?

PSMD11 antibodies enable comprehensive investigation of this protein's contribution to lung adenocarcinoma through multiple experimental approaches:

These approaches collectively provide insights into PSMD11's potential as both a prognostic biomarker and therapeutic target in lung adenocarcinoma .

What insights can single-cell analysis with PSMD11 antibodies provide in cancer research?

Single-cell analysis using PSMD11 antibodies offers unique insights beyond conventional bulk tissue analysis:

• Heterogeneity Characterization:

  • Immunofluorescence with PSMD11 antibodies can reveal expression variations at the single-cell level

  • Allows identification of distinct subpopulations within tumors

  • Helps distinguish between cancer cells, stromal cells, and immune cells

  Researchers have utilized scRNA-seq data from NCBI GEO database (accession code GSE148071) to analyze PSMD11 expression patterns across distinct cell populations .

• Correlation with Cell States:

  • Combine PSMD11 antibody staining with markers of proliferation, stemness, or differentiation

  • Reveals relationships between PSMD11 expression and specific cellular phenotypes

  • Can identify cells undergoing particular processes (e.g., epithelial-mesenchymal transition)

• Spatial Context Analysis:

  • Use multiplex immunofluorescence with PSMD11 and other markers

  • Maintains tissue architecture information

  • Reveals spatial relationships between PSMD11-expressing cells and other cell types

  • Visualized using dimensionality reduction techniques like t-SNE

• Therapy Response Prediction:

  • Compare PSMD11 expression in responding vs. non-responding tumor cells

  • Identify resistant subpopulations based on PSMD11 expression patterns

  • Inform targeted therapy approaches

The combination of PSMD11 antibodies with single-cell technologies provides unprecedented resolution for understanding its role in tumor heterogeneity and progression .

How do PSMD11 expression patterns correlate with immune cell infiltration in tumors?

PSMD11 expression has been significantly correlated with specific immune cell populations, offering insights into tumor immunology:

• Positive Correlations:

  • PSMD11 expression shows strongest positive correlations with:

    • T helper 2 (Th2) cells: Associated with tumor-promoting inflammation

    • Gamma-delta T cells: Context-dependent roles in tumor immunity

    • T regulatory cells (Tregs): Suppress anti-tumor immune responses

  These positive correlations suggest PSMD11 may contribute to immunosuppressive tumor microenvironments .

• Negative Correlations:

  • PSMD11 expression shows strongest negative correlations with:

    • B cells: Important for antibody production and antigen presentation

    • Mast cells: Involved in inflammatory responses

    • CD8+ T cells: Critical for direct tumor cell killing

  These negative correlations suggest that high PSMD11 expression may be associated with reduced anti-tumor immunity .

• MDSC Relationship:

  • PSMD11 expression positively correlates with myeloid-derived suppressor cells (MDSCs)

  • MDSCs are potent immunosuppressive cells that inhibit T cell responses

  • This relationship was investigated using the TIMER platform

  This finding suggests PSMD11 may influence tumor immune evasion through MDSC recruitment or activation .

• Immune Checkpoint Expression:

  • PSMD11 expression correlates with increased expression of immunosuppressive molecules

  • Researchers utilized TCGA database to investigate these associations

  This relationship indicates potential interactions between PSMD11 and immune checkpoint pathways .

These correlations provide mechanistic insights into how PSMD11 may influence the tumor immune microenvironment, potentially guiding immunotherapy approaches .

What emerging techniques might enhance PSMD11 antibody applications in research?

Several cutting-edge technologies are poised to expand the utility of PSMD11 antibodies in research:

• Spatial Transcriptomics and Proteomics:

  • Combining PSMD11 antibody staining with spatial transcriptomics

  • Correlates protein expression with transcriptional profiles while maintaining spatial context

  • Reveals tissue microenvironments where PSMD11 functions most actively

• Proximity Ligation Assays:

  • Detects protein-protein interactions involving PSMD11 in situ

  • Reveals spatial proximity between PSMD11 and other proteasome components or substrates

  • Provides insights into context-specific interaction partners

• Mass Cytometry (CyTOF):

  • Antibodies conjugated to rare earth metals instead of fluorophores

  • Allows simultaneous detection of PSMD11 alongside 40+ other proteins

  • Ideal for comprehensive immune profiling in relation to PSMD11 expression

• Antibody-Based CRISPR Screens:

  • PSMD11 antibodies used to sort cells after CRISPR screens

  • Identifies genes that regulate PSMD11 expression or function

  • Reveals synthetic lethal interactions with PSMD11

• Live-Cell Imaging with Nanobodies:

  • Development of PSMD11-specific nanobodies for live imaging

  • Tracks proteasome dynamics in living cells

  • Reveals real-time changes in PSMD11 localization and function

These emerging technologies promise to provide deeper insights into PSMD11's dynamic functions in normal and disease states .

How might PSMD11 antibodies contribute to therapeutic development for lung adenocarcinoma?

PSMD11 antibodies can facilitate therapeutic development for lung adenocarcinoma through several strategic applications:

• Patient Stratification for Clinical Trials:

  • IHC using PSMD11 antibodies can identify patients with high expression

  • Enables selection of appropriate patients for proteasome-targeting therapies

  • Research has established that PSMD11 expression levels correlate with clinical outcomes in LUAD patients

• Target Validation:

  • Western blot and IHC confirm PSMD11 as a therapeutic target

  • Validates effects of PSMD11 modulation on cancer cell properties

  • Studies have shown that PSMD11 knockdown diminishes proliferation, migration, invasion, and tumor growth in lung carcinoma cell lines

• Companion Diagnostic Development:

  • Standardized PSMD11 antibody-based assays could serve as companion diagnostics

  • Predicts response to proteasome inhibitors or PSMD11-targeted therapies

  • May help identify patients likely to benefit from specific treatment approaches

• Therapeutic Antibody Development:

  • Research-grade antibodies inform development of therapeutic antibodies

  • Potential for antibody-drug conjugates targeting PSMD11-expressing cells

  • May provide selective targeting of cancer cells with high PSMD11 expression

• Monitoring Treatment Response:

  • Serial biopsies analyzed with PSMD11 antibodies

  • Tracks changes in expression during treatment

  • Indicates development of resistance mechanisms

• Combination Therapy Approaches:

  • PSMD11 antibody staining combined with immune markers

  • Informs rational combinations of proteasome inhibitors with immunotherapies

  • Research has shown PSMD11 correlates with immunosuppressive cell populations, suggesting potential synergy with immune checkpoint inhibitors

These applications highlight how PSMD11 antibodies can bridge from basic research to clinical translation in lung adenocarcinoma .