PSMB2 Antibody Pair

What is PSMB2 and why is it significant in research applications?

PSMB2 (Proteasome subunit beta type-2) is a non-catalytic component of the 20S core proteasome complex involved in the proteolytic degradation of most intracellular proteins. The proteasome complex plays numerous essential roles within cells by associating with different regulatory particles. When associated with two 19S regulatory particles, it forms the 26S proteasome and participates in ATP-dependent degradation of ubiquitinated proteins .

PSMB2 is significant in research for several reasons:

• It plays a key role in maintaining protein homeostasis by removing misfolded or damaged proteins

• It has been implicated in various cancers, including glioma, where it correlates with tumor progression

• PSMB2 knockdown has been shown to suppress proteasome activity and affect cell proliferation in cancer models

• It serves as an important biomarker for studying proteasome function in various cellular processes

For researchers, PSMB2 antibodies provide critical tools for investigating proteasome activity and protein degradation pathways across multiple disease models and basic cellular mechanisms.

What are the optimal applications for PSMB2 antibodies in research?

PSMB2 antibodies have been validated across multiple experimental platforms with different optimal applications based on research needs:

For optimal results, researchers should:

• Validate antibody performance in their specific experimental system

• Use positive controls from verified samples (e.g., HEK-293T for WB)

• Optimize antibody dilutions for each application and cell/tissue type

• Consider species cross-reactivity when designing experiments with animal models

How can researchers validate the specificity of PSMB2 antibodies?

Validating antibody specificity is critical for reliable research outcomes. For PSMB2 antibodies, recommended validation methods include:

• Knockdown/Knockout Validation: Several publications have used PSMB2 knockdown models to confirm antibody specificity . This approach provides the strongest evidence for specificity.

• Western Blot Molecular Weight Verification: PSMB2 has a calculated molecular weight of 22-23 kDa. Confirming band appearance at this size helps validate specificity, though researchers should note that the observed MW (23 kDa) sometimes differs slightly from the calculated MW (22 kDa) .

• Multiple Antibody Concordance: Using different antibodies targeting distinct epitopes of PSMB2 that show similar results.

• Recombinant Protein Controls: Many PSMB2 antibodies are raised against recombinant fusion proteins containing sequences corresponding to specific amino acids of human PSMB2 (e.g., AA 1-201 of NP_002785.1) . These recombinants can serve as positive controls.

• Cross-Reactivity Assessment: When studying PSMB2 across species, validate cross-reactivity as documented. For example, some antibodies have verified reactivity with human, mouse, rat, pig, and dog samples .

Properly validated antibodies ensure reliable experimental outcomes and reproducibility across laboratories.

What methodological considerations are important when designing experiments with PSMB2 antibody pairs for ELISA?

When working with PSMB2 antibody pairs for sandwich ELISA, researchers should consider the following methodological guidelines:

• Antibody Pair Selection: For optimal PSMB2 detection, use a combination of capture and detection antibodies that recognize different epitopes. Available commercial pairs typically use rabbit polyclonal antibodies for both capture (unconjugated) and detection (biotin-conjugated) .

• Sample Preparation: PSMB2 has been documented in both cytoplasmic and nuclear localizations . Ensure extraction buffers and protocols effectively solubilize PSMB2 from both compartments.

• Assay Optimization:

  • Optimize coating concentrations of capture antibody

  • Determine optimal sample dilutions

  • Titrate detection antibody concentration

  • Optimize incubation times and temperatures

  • Validate with recombinant PSMB2 protein standard curve

• Cross-reactivity Management: When using antibody pairs with cross-reactivity to multiple species (human, mouse, pig, dog) , validate species-specific detection limits and potential interference from sample matrix components.

• Controls and Normalization: Include positive controls (e.g., cell lysates from HEK-293T, HeLa, or Jurkat cells) that express detectable PSMB2 levels and negative controls (blocking buffer only) in every assay.

• Buffer Compatibility: Most PSMB2 antibody pairs are stored in phosphate buffered solutions (pH 7.4) with stabilizers and glycerol . Ensure assay buffers are compatible with antibody formulation.

How does PSMB2 expression correlate with pathological conditions, and how can antibodies help study these relationships?

Recent research using PSMB2 antibodies has revealed important correlations between PSMB2 expression and various pathological conditions:

• Glioma: PSMB2 plays an oncogenic role in glioma. Studies utilizing PSMB2 antibodies have demonstrated that PSMB2 expression correlates with tumor progression and poor prognosis in glioma patients. Gene Set Enrichment Analysis (GSEA) has linked PSMB2 levels to immune infiltration patterns in glioma tissues .

• Gastric Cancer: Research using PSMB2 antibodies has shown that PSMB2 overexpression promotes proteasome activity, enhances cell proliferation, and suppresses apoptosis in gastric cancer cells. Pharmacological inhibition with MG132 (1 μM) can counteract these effects .

• Hepatocellular Carcinoma: Knockdown of PSMB2 has been shown to suppress hepatocellular carcinoma growth .

• General Cancer Research: PSMB2 is upregulated in various cancer cell lines including ovarian cancer. Antibodies have been instrumental in detecting these expression changes .

• Neurodegenerative Conditions: The upregulation of PSMB2 may indicate activated neuronal defensive mechanisms in vitamin A depletion (VAD) brain regions .

Methodological approaches for studying these relationships include:

• Using antibodies for expression profiling across tissues

• Correlating expression with clinical parameters and survival data

• Combining antibody-based techniques with genetic approaches (knockdown/overexpression)

• Analyzing proteasome activity in relation to PSMB2 expression levels

What are the advanced techniques for using PSMB2 antibodies in cancer research models?

Advanced researchers utilize several sophisticated techniques with PSMB2 antibodies to investigate cancer mechanisms:

• Multi-Parametric Flow Cytometry: PSMB2 antibodies can be incorporated into flow cytometry panels to correlate proteasome function with other cellular markers in cancer cells .

• Proteasome Activity Coupling: Researchers can pair PSMB2 immunodetection with functional proteasome activity assays to understand how structural changes correlate with activity. For example, studies have shown that PSMB2 overexpression directly promotes proteasome activity, while treatment with MG132 dramatically decreases this activity .

• Chemotherapeutic Response Analysis: PSMB2 antibodies help analyze the relationship between PSMB2 expression and response to chemotherapeutics like temozolomide or cisplatin. Ridge regression and "pRRophetic" forecasting have been used to analyze chemotherapy response in samples with varying PSMB2 expression .

• Immune Infiltration Studies: Advanced techniques combine PSMB2 antibody detection with immune cell markers to study how proteasome function affects tumor immune microenvironment. GSEA analysis and Pearson's correlation tests have been used to determine the linkage between immune cell infiltration and PSMB2 mRNA expression .

• Combination with Genetic Manipulation: For mechanistic studies, researchers use PSMB2 antibodies to validate knockdown or overexpression models, as demonstrated in studies where stable PSMB2 knockdown glioma cell lines were established and verified by Western blotting .

• Predictive Biomarker Development: PSMB2 antibodies help develop and validate PSMB2 as a potential predictive biomarker for cancer treatment response, particularly for proteasome inhibitors.

How should researchers troubleshoot inconsistent results when using PSMB2 antibodies?

When encountering inconsistent results with PSMB2 antibodies, researchers should consider these methodological troubleshooting approaches:

• Western Blot Band Size Discrepancies:

  • The observed molecular weight for PSMB2 (23 kDa) sometimes differs slightly from the calculated weight (22 kDa)

  • This is normal and may result from post-translational modifications

  • Different modified forms can produce multiple bands on membranes

• Sample Preparation Issues:

  • PSMB2 has dual localization (cytoplasm and nucleus)

  • Ensure extraction buffers effectively solubilize both compartments

  • Use phosphate buffered solution (pH 7.4) with appropriate detergents for complete extraction

• Antibody Storage and Handling:

  • Store PSMB2 antibodies at -20°C

  • Avoid repeated freeze/thaw cycles

  • Aliquot antibodies for long-term storage

  • Most PSMB2 antibodies are stable for 12 months when properly stored

• Cross-Reactivity Verification:

  • When working across species, verify the documented cross-reactivity

  • Some antibodies work with human, mouse, rat, pig, and dog samples

  • Others may be human-specific or have limited cross-reactivity

• Application-Specific Optimization:

  • Adjust dilutions based on the specific application:

    • WB: 1:500-1:2000

    • IHC: 1:50-1:200

    • IF: 1:50-1:200

  • Titrate antibody concentration for each new sample type

• Protein Modification Considerations:

  • Consider how PSMB2 modifications might affect epitope recognition

  • Use multiple antibodies targeting different regions when possible

How can researchers use PSMB2 antibodies to investigate proteasome function in experimental models?

PSMB2 antibodies provide valuable tools for investigating proteasome function in various experimental settings:

• Proteasome Assembly Studies:

  • PSMB2 is part of the 20S core proteasome structure composed of 4 rings (2 alpha, 2 beta) with 28 non-identical subunits

  • Antibodies can track PSMB2 incorporation into proteasome complexes

  • Co-immunoprecipitation experiments using PSMB2 antibodies can identify interaction partners

• Inhibitor Binding Analysis:

  • PSMB2 may affect inhibitor binding to proteasomes

  • Researchers can use PSMB2 antibodies to investigate structural changes after inhibitor treatment

  • Example: MG132 (1 μM) treatment reduces proteasome activity in cancer cells overexpressing PSMB2

• Functional Manipulation Verification:

  • When establishing PSMB2 knockdown or overexpression models:

    • Confirm expression changes by qPCR and Western blotting

    • Connect expression changes to functional outcomes

  • Studies show PSMB2 knockdown suppresses cell proliferation and invasion in glioma cells

• Cellular Localization:

  • PSMB2 exhibits both cytoplasmic and nuclear localization

  • Immunofluorescence with PSMB2 antibodies can track proteasome distribution

  • Validated in cell lines like MCF-7, HepG2, and PC3

• Proteasome Activity Correlation:

  • PSMB2 overexpression promotes proteasome activity

  • Researchers can correlate PSMB2 protein levels (detected by antibodies) with functional assays of proteasome activity

  • This approach confirmed that PSMB2 overexpression enhances proteasome function in gastric cancer models

• Therapeutic Response Studies:

  • PSMB2 antibodies help track how proteasome composition changes in response to therapeutics

  • This is particularly relevant for cancer research with proteasome inhibitors

What are the important methodological considerations when using PSMB2 antibodies for cell signaling research?

When investigating cell signaling pathways involving PSMB2, researchers should consider these methodological approaches:

• Integration with Signaling Pathway Analysis:

  • PSMB2 and proteasome function affects multiple signaling pathways including:

    • MAPKK activity

    • Epidermal growth factor receptor signaling

    • p53-mediated cell cycle arrest

    • TNF-mediated signaling

    • T cell receptor signaling

  • Use PSMB2 antibodies alongside antibodies for other pathway components

• Cell Cycle Analysis:

  • PSMB2 is involved in G1/S transition of mitotic cell cycle

  • Combine PSMB2 immunodetection with flow cytometry for cell cycle analysis

  • Studies have demonstrated that PSMB2 knockdown affects cell cycle progression in cancer cells

• Co-localization Studies:

  • Use dual immunofluorescence to study PSMB2 co-localization with:

    • Ubiquitinated proteins

    • Other proteasome components

    • Cell cycle regulators

  • This approach has been validated in cell lines like MCF-7

• Apoptosis Pathway Investigation:

  • PSMB2 affects apoptotic pathways

  • Combine PSMB2 detection with apoptosis markers

  • Research shows PSMB2 overexpression suppresses apoptosis in gastric cancer cells

• Temporal Analysis of Signaling Events:

  • Design time-course experiments to track PSMB2 expression changes during signaling activation

  • Use standardized methods for protein extraction and detection

• Inhibitor-Based Approaches:

  • Pair PSMB2 antibody detection with proteasome inhibitor treatments (e.g., MG132)

  • This approach has revealed how proteasome inhibition affects PSMB2-mediated cellular processes

How should researchers interpret PSMB2 expression data in clinical samples?

When analyzing PSMB2 expression in clinical samples using antibody-based techniques, researchers should consider these interpretive guidelines:

• Expression Correlation with Clinical Parameters:

  • Studies using PSMB2 antibodies have linked expression levels to:

    • Patient survival outcomes in glioma

    • Tumor progression markers

    • Response to chemotherapy

  • Use statistical methods like Kaplan-Meier curves, log-rank tests, and Cox models as demonstrated in glioma research

• Comparative Analysis Across Tissue Types:

  • PSMB2 antibodies have been validated in various human tissues:

    • Tumor tissues: Glioma, gastric cancer, hepatocellular carcinoma

    • Normal tissues: Human colon, kidney

  • Always include appropriate normal tissue controls

  • Document expression differences between tumor and adjacent normal tissues

• Integration with Molecular Data:

  • Correlate PSMB2 protein expression with genomic alterations:

    • IDH status

    • 1p/19q codeletion

    • Other molecular markers

  • Use multivariate analyses to determine independent prognostic value

• Immune Context Consideration:

  • PSMB2 levels correlate with immune infiltration patterns

  • When analyzing tumor samples, consider the immune microenvironment

  • GSEA and Pearson's correlation tests help determine linkage between immune cell infiltration and PSMB2 expression

• Quantitative Assessment Methods:

  • Use standardized scoring systems for IHC (e.g., H-score, percentage positive cells)

  • Implement digital pathology approaches for objective quantification

  • Ensure consistent interpretation across observers

• Therapeutic Predictive Value:

  • PSMB2 expression may predict response to proteasome inhibitors

  • When analyzing pre- and post-treatment samples, document expression changes that correlate with response