PSMD11 Antibody

What is PSMD11 and why is it important in cellular biology?

PSMD11 (proteasome 26S subunit, non-ATPase, 11) is a critical component of the 26S proteasome system, a multicatalytic proteinase complex with a highly ordered structure. The 26S proteasome consists of two main parts: a 20S core and a 19S regulator, with PSMD11 serving as a non-ATPase subunit of the 19S regulator (also known as regulatory subunit RPN6) . This complex plays a key role in maintaining protein homeostasis by removing misfolded or damaged proteins and eliminating proteins whose functions are no longer required . The proteasome participates in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair . Specifically, PSMD11 is required for proteasome assembly and plays a key role in increased proteasome activity in embryonic stem cells (ESCs), where its high expression promotes enhanced assembly of the 26S proteasome .

What are the key characteristics of commercially available PSMD11 antibodies?

PSMD11 antibodies are available in multiple formats with different characteristics:

What experimental systems have been validated for PSMD11 antibody use?

PSMD11 antibodies have been validated in various experimental systems:

Cell Lines:

• HeLa cells

• MCF-7 cells

• 293T cells

• U87-MG cells

• PC3 cells

• TF1 cells

• A549 cells (lung carcinoma)

• PC9 cells (lung carcinoma)

Tissues:

• Human lung tissue

• Human stomach cancer tissue

• Rat lung tissue

• Mouse testis tissue

• Rat brain tissue

• Mouse kidney tissue

How should researchers optimize Western blot protocols for PSMD11 detection?

Optimizing Western blot protocols for PSMD11 detection requires careful consideration of several parameters:

Sample Preparation:

• Lyse cells with Laemmli buffer on ice

• For tissue samples, grind in liquid nitrogen and mix with cold protein extraction buffer

• Briefly ultrasonicate and boil samples for 10 minutes

Separation and Transfer:

• Use 10% SDS-PAGE gels for optimal separation

• Transfer to nitrocellulose membranes

Blocking and Antibody Incubation:

• Block membranes with 5% nonfat milk for 1 hour at room temperature

• Dilute primary PSMD11 antibody according to manufacturer recommendations:

  • Typically 1:500-1:2000 for most PSMD11 antibodies

  • For NBP1-30252: 1:250-1:1000

• Incubate with primary antibody overnight at 4°C

• After washing (3× with TBST), incubate with appropriate secondary antibody for 2 hours at room temperature

Detection:

• Use ECL detection system for visualization

• Expected band size is approximately 47 kDa

Controls:

• Positive controls should include HeLa cells, MCF-7 cells, or mouse tissue lysates

What are the critical parameters for successful immunohistochemistry with PSMD11 antibodies?

Successful immunohistochemistry with PSMD11 antibodies requires optimized protocols:

Antigen Retrieval Options:

• Primary recommendation: TE buffer pH 9.0

• Alternative: Citrate buffer pH 6.0

Blocking Protocol:

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

• Block nonspecific binding with 10% normal goat serum (NGS) for 30 minutes

Antibody Application:

• Recommended dilution range: 1:250-1:1000

• Incubate overnight at 4°C for optimal results

Detection System:

• Apply secondary antibody according to manufacturer instructions

• Develop with diaminobenzidine (DAB)

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

What protocol modifications are needed for immunofluorescence detection of PSMD11?

For optimal immunofluorescence detection of PSMD11:

Sample Preparation:

• Culture cells on appropriate coverslips

• Fix according to standard protocols

Antibody Dilution:

• Most PSMD11 antibodies: use at 1:10-1:100 dilution for IF/ICC

• Incubation time: typically overnight at 4°C

Visualization Strategy:

• Counterstain nuclei with DAPI (blue)

• Use species-appropriate fluorophore-conjugated secondary antibodies

  • Example: goat anti-mouse IgG-Alexa fluor 488 (green)

Validated Cell Lines:

• HeLa cells and MCF-7 cells are consistently used for IF validation

How should PSMD11 antibodies be employed for immunoprecipitation studies?

For successful immunoprecipitation of PSMD11:

Antibody Amount:

• Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

Validated Cell Lines:

• MCF-7 cells have been confirmed for successful IP of PSMD11

Applications:

• Co-immunoprecipitation (Co-IP) has been validated with select PSMD11 antibodies

• This technique is valuable for studying protein-protein interactions involving PSMD11

Confirmation:

• Always confirm successful IP by Western blot

• Expected molecular weight of precipitated PSMD11: 47 kDa

What are the most common issues with PSMD11 Western blots and their solutions?

What are the key challenges in immunohistochemistry with PSMD11 antibodies?

Common Challenges and Solutions:

• Poor signal intensity:

  • Try both recommended antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Adjust antibody concentration within recommended range (1:250-1:1000)

  • Extend primary antibody incubation time

• Non-specific background staining:

  • Optimize blocking conditions (increase blocking time)

  • Dilute primary antibody further

  • Include additional washing steps

• Specificity confirmation:

  • Include known positive controls (human lung tissue, rat lung tissue)

  • Include a negative control omitting primary antibody

  • Consider using tissues from knockout models if available

How should researchers address cross-reactivity issues with PSMD11 antibodies?

Cross-reactivity can present significant challenges, particularly with polyclonal antibodies:

Prevention Strategies:

• Select antibodies validated for your specific species of interest

• For mouse tissues using mouse monoclonal antibodies, implement Mouse-on-Mouse blocking steps

• Consider using antibodies raised against species-specific epitopes

Validation Approaches:

• Always include negative controls

• If possible, use PSMD11 knockdown/knockout samples as definitive controls

• Consider multiple antibodies targeting different epitopes to confirm findings

Species Considerations:

• When working with C. elegans, use only antibodies specifically validated for this species

• For cross-species studies, select antibodies with demonstrated multi-species reactivity

How is PSMD11 implicated in cancer research, particularly lung adenocarcinoma?

PSMD11 has emerged as a significant factor in lung adenocarcinoma (LUAD) research:

Expression and Prognostic Value:

• PSMD11 is identified as a critical cuproptosis- and immune-related gene (CIRG) in LUAD

• Patients with low PSMD11 expression displayed improved prognosis compared to those with high expression

• CIRG signature including PSMD11 serves as a reliable, independent prognostic factor

Functional Impact on Cancer Biology:

• Overexpression of PSMD11 enhanced:

  • Proliferation of lung carcinoma cell line A549

  • Migration and invasion capabilities

  • Tumor growth in xenograft models

• Knockdown of PSMD11 diminished these malignant characteristics in lung carcinoma cell line PC9

Immune Microenvironment Implications:

• PSMD11 expression positively correlates with:

  • Infiltration of myeloid-derived suppressor cells (MDSCs)

  • Increased expression of immunosuppressive molecules

• This suggests PSMD11 may influence tumor immune evasion mechanisms

What experimental approaches can be used to study PSMD11's role in proteasome assembly?

Investigating PSMD11's role in proteasome assembly requires sophisticated approaches:

Genetic Manipulation:

• Overexpression systems to study enhanced proteasome assembly

• Knockdown/knockout models using siRNA or CRISPR-Cas9 to assess assembly defects

• These approaches have been successfully employed in lung cancer cell lines (A549, PC9)

Biochemical Analyses:

• Co-immunoprecipitation to identify PSMD11 interaction partners within the proteasome complex

• Proteasome activity assays to assess functional consequences of PSMD11 manipulation

• Sucrose gradient fractionation to analyze proteasome complex integrity

In Vivo Models:

• Xenograft models with modified PSMD11 expression (as described for tumor growth studies)

• Examination of proteasome assembly in various tissues

How does PSMD11 relate to cuproptosis and what experimental methods can investigate this connection?

The emerging connection between PSMD11 and cuproptosis presents exciting research opportunities:

Cuproptosis Context:

• Cuproptosis is a form of regulated cell death induced by copper ions

• It is implicated in mitochondrial metabolism

• PSMD11 has been identified as a cuproptosis-related gene

Experimental Approaches:

• Copper sensitivity assays:

  • Compare copper-induced cell death in PSMD11 overexpression vs. knockdown models

  • Assess mitochondrial function parameters

• Molecular interaction studies:

  • Investigate PSMD11 interaction with known cuproptosis mediators

  • Examine proteasomal degradation of cuproptosis-related proteins

• Expression correlation analyses:

  • Use bioinformatic approaches to analyze correlation between PSMD11 expression and other cuproptosis genes

  • The Cancer Genome Atlas (TCGA) database is a valuable resource for such analyses

What methods can researchers use to investigate the relationship between PSMD11 and immune cell infiltration?

To study PSMD11's relationship with immune cell populations:

Bioinformatic Approaches:

• Tumor Immune Estimation Resource (TIMER) analysis to explore immune cell composition

• Spearman's correlation analyses to describe associations between PSMD11 expression and immune cell proportions

• TCGA database investigations for associations between PSMD11 and immune checkpoints

Experimental Validation:

• Flow cytometry analysis of immune cell populations in models with altered PSMD11 expression

• Immunohistochemistry of tumor tissues to co-localize PSMD11 expression with immune cell markers

• In vitro co-culture systems to study direct interactions between tumor cells expressing PSMD11 and immune cells

Functional Assessment:

• T cell activation assays in the presence of PSMD11-modulated tumor cells

• Analysis of cytokine profiles in the tumor microenvironment

• Investigation of immune checkpoint molecule expression in relation to PSMD11 levels

What are the emerging therapeutic implications of PSMD11 research?

Given its roles in cancer progression and potential immune modulation, PSMD11 presents several therapeutic opportunities:

PSMD11 as a Therapeutic Target:

• Specific inhibitors of PSMD11 might disrupt proteasome assembly in cancer cells

• The observed effects of PSMD11 knockdown on reducing proliferation, migration, and invasion of lung cancer cells suggest therapeutic potential

Biomarker Development:

• PSMD11 expression levels may serve as a valuable prognostic biomarker for LUAD

• Potential predictive biomarker for response to proteasome inhibitors or immunotherapies

Combinatorial Approaches:

• Targeting PSMD11 alongside immune checkpoint inhibitors may enhance efficacy

• Combining PSMD11 modulation with copper-based therapies could exploit the cuproptosis connection

What are the technical challenges in developing more specific tools for PSMD11 research?

Development of more precise PSMD11 research tools faces several challenges:

Antibody Specificity:

• Creating antibodies that distinguish between free PSMD11 and proteasome-incorporated PSMD11

• Developing antibodies specific to post-translationally modified forms of PSMD11

Conditional Models:

• Engineering tissue-specific or inducible PSMD11 knockout/knockin models

• Creating models that specifically disrupt PSMD11's role in proteasome assembly without affecting other functions

Structural Biology Approaches:

• Resolving high-resolution structures of PSMD11 within the proteasome complex

• Identifying specific binding sites for small molecule development

Emerging Technologies:

• Applying proximity labeling approaches to map PSMD11 interactome in different cellular contexts

• Developing CRISPR-based screens to identify synthetic lethal interactions with PSMD11 in cancer contexts