pi049 Antibody

What is PI9/SERPINB9 and what is its biological function?

PI9 (Protease Inhibitor 9) is a 376-amino acid protein that belongs to the Serpin family, specifically the Ov-serpin subfamily. It is encoded by the human gene SERPINB9 and functions primarily as a Granzyme B inhibitor. PI9 is predominantly localized in the cytoplasm where it plays a crucial role in protecting cells from cytotoxic T lymphocyte-mediated apoptosis by inhibiting the serine protease Granzyme B . This protective mechanism is particularly important in immune privilege maintenance and cytotoxic immune response regulation.

What types of anti-PI9 antibodies are commercially available for research?

Multiple types of anti-PI9 antibodies are available for research applications, including:

These antibodies are available from multiple suppliers including Abcam, Biorbyt, Cell Sciences, and Santa Cruz Biotechnology .

What are the common applications of PI9 antibodies in research?

PI9 antibodies are utilized in multiple experimental applications including:

• Western blotting (WB) for protein expression analysis

• Immunohistochemistry on paraffin-embedded tissues (IHC-p)

• Immunocytochemistry (ICC) and immunofluorescence (IF) for cellular localization studies

• Enzyme-linked immunosorbent assay (ELISA) for quantitative analysis

• Immunoprecipitation (IP) for protein complex studies

How should PI9 antibodies be validated before experimental use?

Proper validation of PI9 antibodies should follow a multi-step approach:

• Specificity validation: Test against recombinant human PI9 protein as positive control

• Cross-reactivity testing: Verify specificity against multiple tissue types to confirm minimal non-specific binding

• Application-specific validation:

  • For WB: Verify correct band size (approximately 42 kDa for human PI9)

  • For IHC/ICC: Include appropriate positive control tissues (e.g., human prostate, kidney)

• Knockout/knockdown controls: When possible, validate against samples with reduced or absent PI9 expression

What are the optimal conditions for using anti-PI9 antibodies in Western blotting?

For optimal Western blot results with anti-PI9 antibodies:

• Sample preparation: Use standard cell lysis buffers containing protease inhibitors

• Recommended concentration: 2-3 μg/mL for most commercial antibodies

• Protein loading: 20-30 μg of total protein is typically sufficient

• Expected molecular weight: 42 kDa band for human PI9/SERPINB9

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Secondary antibody selection: Species-appropriate HRP-conjugated secondary antibody (e.g., HRP-linked anti-rabbit or anti-mouse depending on primary antibody)

How can PI9 antibodies be used to study immune evasion in cancer research?

PI9/SERPINB9 antibodies are valuable tools in cancer research, particularly in studying immune evasion mechanisms:

• Expression profiling: Use IHC with anti-PI9 antibodies to assess expression patterns across various cancer types, particularly those with immunotherapy resistance

• Correlation analysis: Combine PI9 immunostaining with clinical outcome data to evaluate prognostic value

• Co-localization studies: Pair PI9 antibodies with immune cell markers (CD8, granzyme B) in multiplexed immunofluorescence to analyze tumor-immune interactions

• Mechanism investigation: Use PI9 antibodies in combination with apoptosis markers to study resistance to cytotoxic T cell-mediated killing

• Therapeutic development: Screen for compounds that modulate PI9 expression as potential immunotherapy enhancers

Several studies have used anti-PI9 antibodies to demonstrate upregulation of PI9 in tumors as a mechanism to escape immune surveillance .

What precautions should be taken when using PI9 antibodies for immunohistochemistry?

When performing immunohistochemistry with PI9 antibodies, consider these critical factors:

• Tissue fixation: Formalin-fixed, paraffin-embedded tissues are suitable, but overfixation may mask epitopes

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically required

• Optimal antibody concentration: Approximately 10 μg/ml for most commercial antibodies on FFPE tissues

• Incubation conditions: Overnight at 4°C or 1-2 hours at room temperature

• Detection system: HRP-linked secondary antibodies (typically 2 μg/ml) with appropriate chromogenic substrate

• Controls: Include known positive tissues (prostate, kidney, bile duct) and negative controls (primary antibody omission)

How does PI9 expression relate to granzyme B inhibition in experimental models?

When investigating the functional relationship between PI9 and granzyme B:

• Co-immunoprecipitation: Use anti-PI9 antibodies to pull down protein complexes and detect granzyme B association

• Cytotoxicity assays: Compare granzyme B-mediated cell death in cells with different PI9 expression levels

• Inhibition kinetics: Use purified recombinant proteins and measure inhibition constants

• Mutagenesis studies: Engineer PI9 variants with modified reactive center loops and assess inhibitory potency

• Cellular localization: Use immunofluorescence with both PI9 and granzyme B antibodies to study subcellular distribution during immune cell attack

Research has shown that PI9 can protect against cytotoxic T lymphocyte-mediated apoptosis by binding and inhibiting granzyme B, which has implications for immune evasion in various diseases .

What are common issues when using PI9 antibodies and how can they be resolved?

How can PI9 antibodies be used in studying autoimmune and inflammatory conditions?

Emerging applications for PI9 antibodies in autoimmune and inflammatory research include:

• Tissue expression analysis: Map PI9 expression in affected tissues from autoimmune disease patients

• Cell-specific expression: Use flow cytometry with anti-PI9 antibodies to identify PI9-expressing immune cell subsets

• Cytokine response: Monitor PI9 expression changes following cytokine stimulation using Western blotting

• Therapeutic biomarker: Evaluate PI9 as a potential biomarker for response to immunotherapies

• Disease mechanisms: Investigate how PI9 expression correlates with disease severity and progression

Studies have suggested that dysregulation of protease inhibitor balance, including PI9, may contribute to pathological immune responses .

What new technologies are enhancing PI9 antibody applications in research?

Recent technological advances are expanding the utility of PI9 antibodies:

• Single-cell analysis: Integration with single-cell technologies to map PI9 expression at single-cell resolution

• Spatial transcriptomics integration: Combining PI9 antibody staining with spatial transcriptomics for correlative analysis

• Multiplexed imaging: Using PI9 antibodies in multiplexed immunofluorescence or mass cytometry panels

• Antibody engineering: Development of recombinant antibodies with improved specificity and reduced lot-to-lot variation

• Chiral optical sensing: Novel detection methods using plasmon-induced chirality transfer are being developed for antibody detection with applications potentially extending to PI9 research

How do PI9/SERPINB9 antibodies contribute to understanding immune checkpoint therapies?

PI9/SERPINB9 antibodies provide valuable insights in checkpoint inhibitor research:

• Resistance mechanism identification: Use PI9 immunostaining to assess whether PI9 upregulation correlates with resistance to immune checkpoint inhibitors

• Combination therapy design: Screen for compounds that modulate PI9 expression as potential synergistic agents with checkpoint inhibitors

• Patient stratification: Evaluate PI9 expression as a potential biomarker for patient selection in immunotherapy trials

• Response monitoring: Track changes in PI9 expression during treatment to monitor adaptive resistance

Research suggests that higher PI9 expression may predict resistance to immunotherapies that rely on cytotoxic T-cell activity .

What considerations should researchers make when selecting between monoclonal and polyclonal PI9 antibodies?

When selecting between monoclonal and polyclonal antibodies, researchers should consider their specific experimental requirements, budget constraints, and the particular application needs .