PSMA1 Antibody, HRP conjugated

What detection applications are PSMA1 antibodies suitable for?

PSMA1 antibodies, including HRP-conjugated versions, have been validated for multiple research applications:

These applications have been verified using high-quality antibodies against PSMA1, with demonstrated reactivity across human, mouse, and rat species .

What are the optimal antigen retrieval methods for PSMA1 detection in tissue sections?

For effective PSMA1 detection in fixed tissues, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has shown optimal results. The validated protocol includes:

• Deparaffinization and rehydration of tissue sections

• Heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Blocking with 10% goat serum to minimize non-specific binding

• Incubation with anti-PSMA1 antibody (2 μg/ml) overnight at 4°C

• Secondary antibody incubation with peroxidase-conjugated anti-rabbit IgG for 30 minutes at 37°C

• Visualization using DAB as the chromogen

This method has been successfully applied to various tissue types including lung adenocarcinoma, rectum adenocarcinoma, urothelial carcinoma, and colon tissues .

How should I design experiments to study PSMA1's role in cancer cell proliferation?

Based on published research methodologies, a comprehensive experimental design should include:

• Cell line selection: Use both PSMA1-high expressing cancer cell lines (e.g., BGC-823, AGS for gastric cancer) and normal control cells (e.g., GES-1)

• Modulation approaches:

  • Knockdown: siRNA transfection targeting PSMA1

  • Overexpression: Transfection with PSMA1 expression plasmids

• Functional assays:

  • Colony formation assay to assess proliferation capacity

  • Transwell migration and invasion assays

  • Cell viability measurements using CCK-8 assay

• Molecular readouts:

  • Expression of proliferation markers (PCNA, C-Myc)

  • Evaluation of downstream effectors (TAZ, YAP)

This approach has successfully demonstrated that PSMA1 suppression decreases viability, migration, and invasion of gastric cancer cells, while overexpression produces the opposite effects .

What controls should be included when using PSMA1 antibodies for cancer tissue staining?

For robust and reliable PSMA1 detection in cancer tissues, include the following controls:

• Positive tissue controls: Include known PSMA1-expressing tissues such as lung adenocarcinoma and rectum adenocarcinoma samples

• Negative controls:

  • Primary antibody omission control

  • Isotype control antibody

  • Normal adjacent tissue when available

• Expression gradient controls: When studying cancer progression, include tissues representing different stages of disease development (e.g., superficial gastritis, intestinal metaplasia, dysplasia, and frank carcinoma)

• Cell line validation: Parallel staining of cell lines with known PSMA1 expression levels

• Antibody concentration titration: Optimize signal-to-noise ratio by testing multiple antibody dilutions

Research has shown progressive increases in PSMA1 expression from superficial gastritis to intestinal metaplasia to dysplasia, with highest levels in gastric cancer tissues, making this gradient valuable for validation .

How can I quantify PSMA1 protein stability and degradation in experimental settings?

To assess PSMA1 protein stability or its effects on substrate proteins like TAZ, implement the following methods:

• Cycloheximide (CHX) chase assay:

  • Treat cells with CHX to inhibit new protein synthesis

  • Harvest cells at multiple time points (0, 2, 4, 8, 12, 24 hours)

  • Analyze protein degradation rates by Western blot

• Proteasome inhibition studies:

  • Treat cells with proteasome inhibitor MG132

  • Compare with autophagy-lysosome inhibition (Chloroquine)

  • Determine if protein degradation occurs via proteasomal pathway

• Ubiquitination assays:

  • Perform co-immunoprecipitation with HA-tagged ubiquitin

  • Test mutant ubiquitin variants to identify specific linkage types (K27, K48, K63)

  • Analyze by Western blot to detect ubiquitinated protein species

These approaches have successfully demonstrated that PSMA1 stabilizes TAZ by removing K27- and K48-linked ubiquitin chains specifically at lysine 214 (K214) of TAZ .

How does PSMA1 interact with the ubiquitin-proteasome system in cancer progression?

PSMA1's relationship with the ubiquitin-proteasome system (UPS) is complex and multifaceted:

• Deubiquitinating activity: Despite being a proteasome component, PSMA1 demonstrates deubiquitinating enzyme (DUB) activity, particularly toward the oncogenic co-activator TAZ

• Specificity of deubiquitination:

  • PSMA1 preferentially removes K27- and K48-linked ubiquitin chains

  • The deubiquitination occurs at specific residues (K214 on TAZ)

  • This selective activity contrasts with its role in the proteasome

• Functional consequences:

  • Stabilization of oncogenic factors increases their half-life

  • Prevention of proteasomal degradation of specific substrates

  • Amplification of downstream signaling pathways

• Mechanistic insights: Co-immunoprecipitation and immunofluorescence studies have confirmed direct physical interaction between PSMA1 and TAZ, with marked colocalization in gastric cancer cells

Understanding this dual role of PSMA1 in both the proteasome and as a deubiquitinating mediator provides significant insights into cancer progression mechanisms and potential therapeutic targeting.

What methodologies are recommended for studying PSMA1-protein interactions?

For investigating PSMA1's interactions with other proteins such as TAZ, employ these methodologies:

• Co-immunoprecipitation (Co-IP):

  • Reciprocal Co-IP using both endogenous proteins and exogenous tagged versions

  • Compare results with native and overexpressed proteins

  • Include appropriate controls (IgG, input lysates)

• Domain mapping:

  • Generate segment plasmids for both PSMA1 and interacting proteins

  • Determine specific interaction domains through truncation mutants

  • Identify critical residues using site-directed mutagenesis

• Immunofluorescence co-localization:

  • Perform dual immunofluorescence staining

  • Analyze colocalization using confocal microscopy

  • Quantify colocalization using appropriate software

• Proximity ligation assay (PLA):

  • For detecting protein interactions in situ with high sensitivity

  • Particularly valuable for detecting transient interactions

• Proteomic screening:

  • TMT labeling experiments to systematically detect protein expression changes

  • Compare PSMA1-knockdown vs. control cells to identify potential substrates

These methods have successfully identified TAZ as a key interacting partner of PSMA1, with 34 downregulated and 86 upregulated proteins detected using TMT labeling after PSMA1 knockdown .

How can researchers translate PSMA1 findings from cell lines to clinical significance?

To establish clinical relevance of PSMA1 research, implement this progression from bench to bedside:

• Patient sample analysis:

  • Compare PSMA1 expression in tumor tissues versus matched normal tissues

  • Analyze expression across disease progression stages

  • Correlate with clinical parameters (tumor stage, lymph node metastasis, survival)

• Clinical correlation studies:

  • Create tissue microarrays (TMAs) from patient cohorts

  • Perform IHC to quantify PSMA1 expression

  • Use Kaplan-Meier survival analysis to determine prognostic significance

• Multivariate analysis:

  • Assess if PSMA1 is an independent prognostic factor

  • Include standard clinical parameters in the analysis

  • Determine hazard ratios for high vs. low expression

• Validation in multiple cohorts:

  • Confirm findings in independent patient cohorts

  • Validate using public databases (TCGA, GEO)

  • Conduct meta-analysis when possible

Research has demonstrated that high PSMA1 expression significantly correlates with advanced tumor stage (p = 0.006) and positive lymph node metastasis (p = 0.041), with Kaplan-Meier analysis showing worse prognosis in patients with high PSMA1 expression .

What are the most common issues when using HRP-conjugated PSMA1 antibodies and how can they be resolved?

When working with HRP-conjugated PSMA1 antibodies, researchers may encounter these challenges:

For immunohistochemistry applications specifically, the protocol validated for PSMA1 detection recommends overnight incubation at 4°C with 2 μg/ml antibody concentration following heat-mediated antigen retrieval in EDTA buffer (pH 8.0) .

How should researchers validate the specificity of PSMA1 antibodies in their experimental systems?

Comprehensive validation of PSMA1 antibodies should include:

• Knockdown/knockout validation:

  • Use siRNA to suppress PSMA1 expression

  • Verify reduction of signal in Western blot and IHC

  • Include appropriate non-targeting controls

• Overexpression validation:

  • Transfect cells with PSMA1 expression constructs

  • Confirm increased signal intensity in Western blot

  • Verify subcellular localization in immunofluorescence

• Cross-species reactivity testing:

  • Test antibody in human, mouse, and rat samples

  • Compare staining patterns across species

  • Ensure consistent results in analogous tissues

• Peptide competition assay:

  • Pre-incubate antibody with blocking peptide

  • Confirm signal reduction or elimination

  • Use as negative control in parallel experiments

• Multiple antibody validation:

  • Compare results using antibodies targeting different PSMA1 epitopes

  • Ensure consistent detection patterns

These validation steps are essential for confirming that observed signals truly represent PSMA1 expression and not artifacts or cross-reactivity with other proteins.

What emerging research areas involving PSMA1 should investigators consider exploring?

Based on current findings, these promising research directions warrant further investigation:

• PSMA1 as a therapeutic target:

  • Development of specific PSMA1 inhibitors that modulate its deubiquitinating activity

  • Exploration of synthetic lethality approaches in PSMA1-high cancers

  • Investigation of combination therapies targeting PSMA1 and its downstream effectors

• PSMA1 as a biomarker:

  • Validation of PSMA1 as a prognostic biomarker across multiple cancer types

  • Development of PSMA1-based liquid biopsy approaches

  • Correlation of PSMA1 expression with treatment response

• PSMA1 biology:

  • Further characterization of PSMA1's dual role in proteasome function and deubiquitination

  • Identification of additional PSMA1 substrates beyond TAZ

  • Investigation of PSMA1's role in cancer stem cell maintenance and therapy resistance

• Translational applications:

  • Development of PSMA1-targeted imaging approaches for cancer detection

  • Exploration of PSMA1 as an immunotherapy target

  • Creation of PSMA1-based chimeric antigen receptor (CAR) T-cell therapies

The demonstration that PSMA1 promotes gastric cancer progression through deubiquitination of TAZ opens numerous avenues for therapeutic development and further mechanistic studies .

How might researchers investigate the relationship between PSMA1 and response to proteasome inhibitor therapies?

To explore PSMA1's impact on proteasome inhibitor efficacy, consider these research approaches:

• Expression correlation studies:

  • Compare PSMA1 expression levels across cell lines with varying proteasome inhibitor sensitivity

  • Analyze patient data for correlations between PSMA1 expression and clinical response

  • Perform meta-analysis of existing clinical trial data

• Mechanistic investigations:

  • Modulate PSMA1 expression (knockdown/overexpression) and assess changes in proteasome inhibitor sensitivity

  • Investigate whether PSMA1's deubiquitinating activity contributes to proteasome inhibitor resistance

  • Determine if PSMA1 inhibition synergizes with proteasome inhibitors

• Development of combination approaches:

  • Test combining proteasome inhibitors with agents targeting TAZ or other PSMA1 substrates

  • Investigate sequential treatment strategies

  • Explore synthetic lethality approaches

• Biomarker development:

  • Assess if PSMA1 expression or activity can predict response to proteasome inhibitors

  • Develop assays to measure PSMA1-specific deubiquitinating activity in patient samples

  • Correlate with treatment outcomes in clinical studies

These investigations could potentially identify patient populations most likely to benefit from proteasome inhibitor therapies and suggest rational combination strategies to enhance efficacy.