PSMC6 Antibody

What is PSMC6 and why is it significant in biological research?

PSMC6 (proteasome 26S subunit, ATPase, 6) is a critical component of the 26S proteasome complex involved in ATP-dependent degradation of ubiquitinated proteins. Also known as SUG2, CADP44, p42, or P44, it belongs to the AAA ATPase family and plays essential roles in protein homeostasis, cell cycle progression, apoptosis, and DNA damage repair . PSMC6 forms part of the heterohexameric ring of AAA proteins that unfolds ubiquitinated target proteins for translocation into the proteolytic chamber for degradation into peptides .

The significance of PSMC6 extends beyond basic cellular functions to disease mechanisms. Research has demonstrated that PSMC6 deficiency correlates with bortezomib resistance in multiple myeloma, making it a crucial target for cancer research . Additionally, PSMC6 overexpression has been associated with poor prognosis in lung adenocarcinoma and breast cancer, highlighting its potential as a prognostic biomarker .

For optimal preservation of PSMC6 antibody activity, the following storage protocol is recommended:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• Most commercial PSMC6 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) or similar buffer systems

• Antibodies are typically stable for one year after shipment when stored properly

• Small volume antibodies (e.g., 20μl sizes) often contain 0.1% BSA for additional stability

• Avoid repeated freeze-thaw cycles by preparing working aliquots upon receipt

When working with PSMC6 antibodies, always handle according to standard laboratory practices for protein-based reagents. The presence of glycerol in the storage buffer helps prevent freezing damage to the antibody structure, while sodium azide prevents microbial contamination .

How can I validate the specificity of my PSMC6 antibody for my experimental system?

Validating PSMC6 antibody specificity requires a multi-step approach:

• Positive tissue controls: Confirmed positive tissues for PSMC6 antibody reactivity include mouse brain, human brain, human testis, rat liver, mouse liver, and rat brain tissues . Include at least one validated positive control in your experiment.

• Knockdown/knockout validation: Generate PSMC6 knockdown cells using CRISPR-Cas9 or siRNA approaches. A validated antibody should show reduced or absent signal in these cells compared to wild-type . In published studies, PSMC6 deficiency was confirmed via reduction in protein levels detected by specific antibodies .

• Molecular weight verification: PSMC6 has a calculated molecular weight of 44 kDa (389 amino acids), but typically appears at approximately 42 kDa on Western blots . Verify that your detected band matches this expected size.

• Cross-reactivity assessment: Test the antibody in multiple species if cross-reactivity is claimed. Most PSMC6 antibodies show reactivity with human, mouse, and rat samples, but specific epitope recognition may vary .

• Immunogen mapping: Confirm that the antibody's immunogen sequence (commonly amino acids 20-150 or the internal region of human PSMC6) is conserved in your experimental species .

What are the optimal antigen retrieval methods for PSMC6 immunohistochemistry?

For optimal PSMC6 detection in immunohistochemistry, the following antigen retrieval protocols have been validated:

• Primary recommendation: TE buffer pH 9.0 has demonstrated superior results for PSMC6 antigen retrieval in tissues including human liver cancer tissue and mouse brain tissue .

• Alternative method: Citrate buffer pH 6.0 can also be used effectively as an alternative retrieval solution .

• Protocol optimization: Regardless of buffer choice, perform heat-induced epitope retrieval (HIER) using either:

  • Pressure cooker method (recommended): 3-5 minutes at full pressure

  • Microwave method: 15-20 minutes at medium power in retrieval buffer

  • Water bath: 30-40 minutes at 95-98°C

• Tissue-specific considerations: Formalin-fixed paraffin-embedded (FFPE) tissues typically require more rigorous retrieval than frozen sections. The duration of fixation can significantly impact retrieval efficacy, with longer fixation times requiring more intensive retrieval .

After antigen retrieval, allow sections to cool to room temperature before proceeding with immunostaining. For PSMC6 antibodies, typical IHC dilutions range from 1:250 to 1:1000, but should be optimized for each specific antibody and tissue type .

How can I optimize Western blot conditions for detecting PSMC6 in different sample types?

Optimizing Western blot conditions for PSMC6 detection requires attention to several key parameters:

• Sample preparation:

  • For tissue lysates: Homogenize tissues in RIPA buffer containing protease inhibitors

  • For cell lysates: Use mild detergent-based lysis buffers (e.g., NP-40 or RIPA)

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Recommended positive control tissues: mouse/human brain, human testis, rat/mouse liver

• Protein loading and separation:

  • Load 20-50 μg total protein per lane

  • Use 10-12% polyacrylamide gels for optimal separation

  • PSMC6 migrates at approximately 42 kDa (calculated MW: 44 kDa)

• Transfer and blocking conditions:

  • Semi-dry or wet transfer systems are suitable

  • PVDF membranes generally provide better results than nitrocellulose

  • Block in 5% non-fat milk or BSA in TBST (depending on antibody specifications)

• Antibody dilution ranges:

  • Primary antibody: Start with 1:1000 dilution (ranges from 1:1000-1:8000 reported)

  • Secondary antibody: Typically 1:5000-1:10000 dilution of HRP-conjugated anti-rabbit IgG

• Detection optimization:

  • Enhanced chemiluminescence (ECL) is sufficient for most applications

  • For low abundance samples, consider using more sensitive ECL substrates

  • Exposure times typically range from 30 seconds to 5 minutes

Researchers should empirically determine the optimal conditions for their specific sample types and antibodies, as PSMC6 expression levels vary across tissues and experimental conditions .

How can PSMC6 antibodies be used to investigate cancer biology and drug resistance mechanisms?

PSMC6 antibodies provide powerful tools for investigating cancer biology and drug resistance through several advanced approaches:

• Bortezomib resistance screening: CRISPR genome-wide screening identified PSMC6 as a key determinant of bortezomib sensitivity in multiple myeloma. PSMC6 antibodies can be used to confirm PSMC6 knockdown and correlate protein levels with drug resistance phenotypes . This approach revealed that PSMC6 deficiency significantly reduces bortezomib-induced inhibition of chymotrypsin-like proteasome activity .

• Prognostic biomarker assessment: In lung adenocarcinoma (LUAD) and breast cancer, PSMC6 overexpression correlates with poor prognosis. PSMC6 antibodies enable IHC analysis of tumor tissues to establish expression patterns and potential prognostic value .

• Immune infiltration profiling: PSMC6 expression correlates with distinct immune cell infiltration patterns in tumors. Research has shown relationships between PSMC6 expression and increased M1 macrophages but decreased B-cell abundance in lung adenocarcinoma . IHC or multiplexed IF using PSMC6 antibodies alongside immune cell markers can map these relationships in tumor microenvironments.

• Therapeutic target validation: PSMC6-directed therapies can be monitored using antibodies to confirm target engagement. In studies where PSMC6 was suppressed to reduce growth and metastasis of LUAD cells, antibodies provided crucial validation of the knockdown phenotype .

• Correlation with immune checkpoint expression: PSMC6 expression positively correlates with immune checkpoint genes, including CD276 (B7-H3), PVR (CD155), and TNFRSF12A . Co-staining with PSMC6 antibodies and checkpoint protein antibodies can reveal spatial relationships in tumor tissues.

What approaches can resolve contradictory PSMC6 expression data in different cancer types?

Resolving contradictory PSMC6 expression data across cancer types requires systematic analytical approaches:

• Standardized quantification methods:

  • Implement consistent Western blot quantification protocols with appropriate loading controls

  • Use automated digital pathology scoring for IHC to minimize subjective interpretation

  • Establish calibrated positive controls across experiments for normalization

• Multi-omics integration strategy:

  • Correlate protein expression (by antibody-based methods) with mRNA expression data

  • Analyze protein levels in context of genomic alterations (mutations, copy number)

  • Example: In lung adenocarcinoma, PSMC6 mRNA overexpression correlates with worse survival outcomes and genomic instability features

• Subcellular localization analysis:

  • Use fractionation followed by Western blot or immunofluorescence with PSMC6 antibodies

  • Determine if contradictory results stem from differences in subcellular distribution

  • Cancer-specific redistribution may explain functional differences despite similar total expression

• Context-dependent modifiers:

  • Evaluate PSMC6 in relation to other PSMC family members

  • Analyze correlation patterns between PSMC genes (example: PSMC1 correlations with PSMC2-6 range from R=0.11 to R=0.42, p<0.05)

  • Consider tissue-specific regulatory factors that may modify PSMC6 function

• Cell-type specific expression patterns:

  • Use single-cell approaches with PSMC6 antibodies to resolve cell-type specific expression

  • In heterogeneous tumors, bulk analysis may mask important cell subpopulation differences

  • Correlate with immune infiltration profiles which may vary by cancer type

How can PSMC6 antibodies be incorporated into CRISPR-based functional genomics workflows?

PSMC6 antibodies serve as essential validation tools in CRISPR-based functional genomics workflows through the following integrated approaches:

• Knockout validation protocol:

  • Design sgRNAs targeting PSMC6 (validated examples from published studies available)

  • Transfect cells with Cas9 and sgRNAs using appropriate delivery method

  • Confirm knockout efficiency using PSMC6 antibodies via Western blot (1:1000-1:2000 dilution)

  • Expected result: Complete absence or significant reduction of the 42 kDa PSMC6 band

• Phenotypic screening correlation:

  • In genome-wide CRISPR screens focusing on proteasome function or drug resistance

  • Use PSMC6 antibodies to validate hits from screens

  • Example application: CRISPR screening of 19,052 human genes identified PSMC6 as the key determinant of bortezomib sensitivity in multiple myeloma cells

• Mechanistic pathway analysis:

  • After CRISPR-mediated PSMC6 deletion, use antibodies to assess effects on:

    • Other proteasome components

    • Ubiquitinated protein accumulation

    • Cell cycle regulatory proteins

  • This approach revealed that PSMC6 deficiency significantly reduced the inhibition of chymotrypsin-like proteasome activity by bortezomib

• Multi-gene interaction studies:

  • Apply CRISPR to create combinatorial knockouts of PSMC family members

  • Use antibodies specific to each PSMC protein to confirm knockouts

  • Published data shows that deficiency in each PSMC subunit (PSMC1-6) imparts bortezomib resistance

  • Antibody-based validation is crucial to confirm the specific contributions of each subunit

• Clinical correlation potential:

  • Link CRISPR-generated phenotypes to patient samples

  • Use PSMC6 antibodies to analyze expression in patient-derived materials

  • In multiple myeloma, 36 mutations in 19S proteasome subunits were found in 895 patients, potentially affecting PSMC6 expression and function

What are common technical issues with PSMC6 antibodies and how can they be resolved?

Researchers commonly encounter several technical challenges when working with PSMC6 antibodies. Here are systematic approaches to resolve these issues:

• Weak or absent Western blot signal:

  • Problem diagnosis: Insufficient protein, degraded antibody, or inadequate transfer

  • Resolution approach:

    • Increase protein loading (40-50 μg recommended)

    • Verify antibody quality with a positive control (mouse/human brain tissue recommended)

    • Optimize primary antibody concentration (try 1:500 instead of 1:1000)

    • Increase incubation time to overnight at 4°C

    • Use more sensitive detection reagents

• Multiple bands on Western blot:

  • Problem diagnosis: Non-specific binding, degradation products, or isoforms

  • Resolution approach:

    • Increase blocking stringency (5% BSA instead of milk)

    • Verify expected molecular weight (PSMC6 appears at ~42 kDa)

    • Include protease inhibitors during sample preparation

    • Increase washing time and volume

    • Consider alternative antibody clones if available

• High background in immunohistochemistry:

  • Problem diagnosis: Insufficient blocking, non-specific binding, or over-development

  • Resolution approach:

    • Optimize antigen retrieval (TE buffer pH 9.0 recommended for PSMC6)

    • Increase antibody dilution (1:500-1:1000 range)

    • Include additional blocking steps (avidin/biotin block if using biotin-based detection)

    • Reduce DAB development time

    • Perform peptide competition assays to confirm specificity

• Variability between experiments:

  • Problem diagnosis: Inconsistent technique, antibody degradation, or sample variation

  • Resolution approach:

    • Aliquot antibody upon receipt to avoid freeze-thaw cycles

    • Standardize protocols including incubation times and temperatures

    • Include internal reference controls in each experiment

    • Document lot numbers and dates for all reagents

• Species cross-reactivity issues:

  • Problem diagnosis: Epitope differences between species or non-specific binding

  • Resolution approach:

    • Verify the immunogen sequence conservation in your species of interest

    • Select antibodies raised against conserved regions (amino acids 20-150 recommended)

    • Validate with species-specific positive controls

    • Consider species-specific secondary antibodies to reduce background

How can I assess PSMC6 antibody lot-to-lot variation to ensure experimental reproducibility?

Maintaining experimental reproducibility through antibody lot changes requires systematic quality control measures:

• Establish reference standard protocol:

  • Create a panel of control samples with known PSMC6 expression levels

  • Recommended controls: mouse brain tissue, human brain tissue, and rat liver tissue

  • Process and store aliquots at -80°C to minimize freeze-thaw cycles

  • Document expected band intensity and pattern for each control

• Quantitative comparison methodology:

  • When receiving a new antibody lot:

    • Run side-by-side Western blots with old and new lots

    • Use identical samples, loading amounts, and development times

    • Quantify signal intensity using digital imaging software

    • Calculate correlation coefficient between measurements

    • Accept only if correlation exceeds 0.9 (R²>0.9)

• Titration curve validation:

  • Perform serial dilutions (1:500, 1:1000, 1:2000, 1:4000, 1:8000) with both lots

  • Plot signal intensity vs. dilution for each lot

  • Compare curve shapes and EC50 values

  • Acceptable variation: EC50 within ±20% between lots

• Cross-application verification:

  • If using the antibody for multiple applications (WB, IHC, IF)

  • Validate each application separately with the new lot

  • Document optimal dilutions for each application

  • Note application-specific differences between lots

• Epitope mapping confirmation:

  • If resources permit, perform epitope mapping on critical antibody lots

  • Use peptide arrays covering the PSMC6 sequence (focus on amino acids 20-150)

  • Compare binding patterns between lots

  • Document any shift in epitope recognition

How does sample preparation affect PSMC6 antibody detection in different experimental contexts?

Sample preparation significantly impacts PSMC6 antibody detection across experimental platforms. Here's a methodological approach to optimize detection in various contexts:

• Western blot sample preparation:

  • Lysis buffer selection effects:

    • RIPA buffer: Good for general PSMC6 detection

    • NP-40 buffer: Preserves protein-protein interactions, beneficial for co-immunoprecipitation

    • SDS lysis buffer: Strongest extraction but may denature epitopes

  • Sample processing impact:

    • Mechanical disruption (sonication/homogenization) improves PSMC6 extraction

    • Centrifugation speed affects yield (10,000-14,000g for 10-15 minutes recommended)

    • Temperature sensitivity: Prepare samples on ice to prevent degradation

• Immunohistochemistry preparation factors:

  • Fixation parameters:

    • Fixative type: 10% neutral buffered formalin recommended for PSMC6

    • Fixation duration: 24-48 hours optimal (over-fixation reduces antibody binding)

    • Post-fixation processing: Minimize dehydration time to prevent antigen masking

  • Antigen retrieval critical factors:

    • Buffer selection: TE buffer pH 9.0 superior for PSMC6 epitope retrieval

    • Alternative method: Citrate buffer pH 6.0 acceptable with extended retrieval time

    • Temperature/time relationship: Higher temperature reduces required retrieval time

• Immunofluorescence-specific considerations:

  • Fixation method comparison:

    • Paraformaldehyde (4%): Preserves PSMC6 subcellular localization

    • Methanol: Better for nuclear PSMC6 but disrupts membrane structures

    • Mixed fixation: PFA followed by methanol permeabilization improves nuclear PSMC6 detection

  • Permeabilization effect on detection:

    • Triton X-100 (0.1-0.5%): Standard for PSMC6 detection

    • Saponin (0.1%): Gentler alternative for detecting membrane-associated PSMC6

    • Digitonin (50 μg/ml): Selective permeabilization useful for distinguishing cytosolic from organelle-associated PSMC6

• Flow cytometry preparation requirements:

  • Cell fixation methods:

    • Paraformaldehyde (1-2%): Maintains epitope accessibility

    • Fixation time: 10-15 minutes at room temperature optimal

  • Permeabilization optimization:

    • Methanol (-20°C): Excellent for intracellular PSMC6 detection

    • Saponin (0.1%): Reversible permeabilization for better epitope access

    • Buffer composition: PBS with 2% BSA reduces non-specific binding

• Chromatin immunoprecipitation (ChIP) considerations:

  • Crosslinking parameters:

    • Formaldehyde concentration: 1% for 10 minutes optimal

    • Quenching method: Glycine (125 mM) for 5 minutes

  • Sonication protocol:

    • Fragment size: 200-500 bp optimal for PSMC6 ChIP

    • Sonication buffer: SDS-containing buffers improve chromatin solubilization

    • Pre-clearing step: Critical for reducing background in PSMC6 ChIP experiments

How are PSMC6 antibodies being used to investigate the relationship between proteasome function and cancer immunotherapy response?

PSMC6 antibodies are providing critical insights into the emerging relationship between proteasome function and immunotherapy response through several advanced research approaches:

• Correlation of PSMC6 expression with immune checkpoint molecules:

  • PSMC6 expression positively correlates with immune checkpoint genes including CD276 (B7-H3), PVR (CD155), and TNFRSF12A in lung adenocarcinoma

  • Methodological approach: Multiplex immunofluorescence using PSMC6 antibodies alongside checkpoint antibodies to map spatial relationships within tumor microenvironments

  • This reveals potential mechanistic links between proteasome function and checkpoint regulation

• Assessment of T-cell dysfunction and exclusion (TIDE):

  • PSMC6 expression correlates with the TIDE score, indicating an immune profile with dysfunction or exclusion of T cells

  • Research methodology: Combine PSMC6 antibody-based tissue analysis with computational algorithms to predict immunotherapy response

  • Finding: High PSMC6 expression associates with immune cold features in lung adenocarcinoma, potentially reducing immunotherapy efficacy

• Mapping tumor-infiltrating immune cell profiles:

  • PSMC6 expression correlates with distinct patterns of immune cell infiltration:

    • Increased M1 macrophage presence

    • Exclusion of memory and plasma B cells

  • Technical approach: Co-staining with PSMC6 and immune cell marker antibodies

  • Result: This provides a potential mechanism for PSMC6's impact on tumor immunity

• Prediction of immunotherapy response:

  • High PSMC6 risk scores correlate with decreased sensitivity to immunotherapy but increased response to platinum-based chemotherapy

  • Method: Stratify patients using PSMC6 antibody-based expression analysis prior to therapy

  • Application: Potential biomarker for therapy selection in lung adenocarcinoma

• Investigation of antigen presentation machinery:

  • PSMC6 as part of the proteasome regulates antigen processing for MHC presentation

  • Experimental approach: Analyze correlation between PSMC6 expression and MHC class I levels using antibody-based techniques

  • Finding: Alterations in PSMC6 may impact antigen presentation efficiency, affecting T cell recognition

This emerging research direction suggests that PSMC6 antibodies may become important tools in predicting and understanding immunotherapy response, potentially guiding therapeutic decisions for cancer patients.

What are the emerging applications of PSMC6 antibodies in studying neurodegenerative diseases?

PSMC6 antibodies are increasingly utilized in neurodegenerative disease research, revealing important connections between proteasome dysfunction and neurodegeneration:

• Proteasome impairment assessment in neural tissues:

  • PSMC6 antibodies detect alterations in proteasome composition in neurodegenerative conditions

  • Methodology: Compare PSMC6 levels in affected vs. unaffected brain regions using IHC and Western blot

  • Brain tissues (mouse and human) are established positive controls for PSMC6 antibodies

  • Application: Quantitative assessment of proteasome dysfunction in Alzheimer's, Parkinson's, and ALS models

• Protein aggregation relationship studies:

  • Investigate correlation between PSMC6 levels and protein aggregates

  • Technical approach: Co-localization studies using PSMC6 antibodies with aggregate markers

  • Dual-label immunofluorescence with PSMC6 (1:250-1:500) and aggregate markers

  • Finding: Altered PSMC6 distribution in proximity to aggregation-prone proteins

• Stress granule association analysis:

  • PSMC6 may interact with stress granule components in neurodegenerative conditions

  • Research method: Immunoprecipitation with PSMC6 antibodies followed by proteomic analysis

  • Co-immunostaining for PSMC6 and stress granule markers

  • Significance: Links between proteasome function and RNA metabolism in neurodegeneration

• Neuroinflammation correlation studies:

  • PSMC6 expression changes in response to neuroinflammatory conditions

  • Approach: Analyze PSMC6 levels in microglia and astrocytes using cell-type specific markers and PSMC6 antibodies

  • Compare expression in activated vs. resting glial cells

  • Result: Potential biomarker for neuroinflammatory component of neurodegenerative diseases

• Therapeutic response monitoring:

  • PSMC6 antibodies to track proteasome function during experimental treatments

  • Method: Longitudinal assessment of PSMC6 levels and localization before and after treatment

  • Western blot quantification with normalization to housekeeping proteins

  • Application: Biomarker for therapeutic efficacy of proteasome-targeting drugs in neurodegenerative diseases

These emerging applications position PSMC6 antibodies as valuable tools in understanding the complex relationship between protein homeostasis and neurodegeneration, potentially leading to new therapeutic approaches.

How can high-throughput approaches incorporate PSMC6 antibody-based detection for biomarker development?

High-throughput approaches utilizing PSMC6 antibody-based detection offer promising avenues for biomarker development:

• Tissue microarray (TMA) screening methodology:

  • Create TMAs from multiple patient cohorts representing different cancer types

  • Apply standardized IHC protocol using validated PSMC6 antibodies (1:250-1:1000 dilution)

  • Implement digital pathology scoring with machine learning algorithms

  • Results from previous studies: PSMC6 overexpression correlates with poor prognosis in lung adenocarcinoma and breast cancer

  • Output: Quantitative PSMC6 expression data across hundreds of samples simultaneously

• Reverse phase protein array (RPPA) implementation:

  • Prepare lysates from patient samples or cell lines

  • Spot in array format on nitrocellulose-coated slides

  • Probe with validated PSMC6 antibodies

  • Develop fluorescent or chromogenic detection system

  • Data analysis: Correlate PSMC6 levels with clinical parameters

  • Application: Rapidly screen large cohorts for PSMC6 as a drug resistance biomarker in multiple myeloma

• Multiplex immunofluorescence panel development:

  • Design panels including PSMC6 alongside immune markers (based on correlations with immune checkpoint genes)

  • Optimize antibody combination with PSMC6 (1:500) and checkpoint markers

  • Apply tyramide signal amplification for increased sensitivity

  • Analyze using multispectral imaging platforms

  • Finding: PSMC6 expression correlates with specific immune profiles that may predict therapy response

• Automated Western blot platform adaptation:

  • Implement PSMC6 detection on capillary-based Western systems

  • Optimize antibody concentration (typically 1:1000-1:2000)

  • Develop automated analysis parameters for PSMC6 quantification

  • Validate against traditional Western blot results

  • Output: High-throughput quantitative data on PSMC6 protein levels across sample sets

• Mass cytometry (CyTOF) integration:

  • Conjugate PSMC6 antibodies with rare earth metals

  • Incorporate into panels with cell type markers and functional readouts

  • Apply to dissociated tissue samples or blood cells

  • Analyze using dimensional reduction and clustering algorithms

  • Application: Correlate PSMC6 levels with cell state and function at single-cell resolution

  • Finding: Link between PSMC6 expression, cellular phenotype, and response to proteasome inhibitors