PBL9 Antibody

What is PI-9 (SERPINB9) and what is its primary function?

PI-9, also known as SERPINB9, is a serine protease inhibitor that functions primarily as a granzyme B inhibitor. It acts as part of a complex that manages cell death pathways, serving as a guardian against excessive cytotoxic actions by the immune system. The protein's interaction with granzyme B highlights its role in maintaining the balance between immune defense and cellular protection .

The protein is also known by several other names including Serpin B9, Cytoplasmic antiproteinase 3, Peptidase inhibitor 9, CAP-3, and CAP3. As a member of the serpin superfamily, PI-9 plays a crucial role in regulating proteolytic cascades involved in various physiological processes including inflammation, blood coagulation, and programmed cell death.

PI-9's protective function is particularly important in cells that contain cytotoxic granules themselves, helping to prevent self-induced apoptosis in immune effector cells like cytotoxic T lymphocytes and natural killer cells.

What are the common applications for PI-9 antibody in research?

PI-9 antibodies are suitable for multiple experimental applications including Western blot (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) . These methods allow researchers to detect and quantify PI-9 expression in various cell and tissue types.

In Western blotting, PI-9 antibody can detect the protein with a predicted band size of approximately 42 kDa in various samples including human serum, mouse placenta lysate, and cell lines such as K562 (human chronic myelogenous leukemia cell line) . For immunohistochemistry, the antibody can be used to study PI-9 expression in various tissues including prostate, kidney, cerebrum, stomach, and cancer tissues like bile duct cancer and prostate cancer .

Additionally, PI-9 antibodies are valuable tools for investigating the role of this protein in regulating cytotoxic T lymphocyte and natural killer cell-mediated apoptosis, and its potential role in tumor immune evasion mechanisms.

What tissue types typically express PI-9 and how is this detected?

PI-9 expression has been detected in multiple human tissue types through immunohistochemical analysis. Based on the search results, PI-9 expression has been observed in bile duct cancer tissue, prostate tissue (both normal and cancerous), kidney tissue, cerebrum, and stomach tissue .

To detect PI-9 in these tissues, researchers typically use techniques like immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue sections. The protocol generally involves using the PI-9 antibody at a concentration of approximately 10 μg/ml followed by detection with an appropriate secondary antibody, such as HRP-Linked Caprine Anti-Rabbit IgG Polyclonal Antibody (typically at 2 μg/ml) .

The expression pattern of PI-9 across different tissues provides valuable insights into the physiological roles of this protein in various organ systems and may help identify tissues where protection against granzyme B-mediated apoptosis is particularly important. Variations in expression levels between normal and pathological tissues can also provide insights into disease mechanisms.

How should researchers validate the specificity of PI-9 antibody for their experimental systems?

Antibody validation is critical for ensuring experimental reproducibility and generating reliable data. For PI-9 antibody, researchers should implement a multi-pronged validation approach that includes both positive and negative controls.

Positive controls should include tissues or cell lines known to express PI-9, such as K562 cells or human placenta tissue . Western blotting with recombinant human PI-9 protein can confirm antibody specificity by demonstrating binding to a protein of the expected molecular weight (42 kDa) . Researchers should also verify that the antibody detects the target in appropriate cellular compartments, as PI-9 is primarily cytoplasmic.

Negative controls are equally important and should include samples known not to express PI-9 or samples where PI-9 expression has been knocked down using siRNA or CRISPR-Cas9 techniques. As emphasized in the literature on antibody characterization, approximately 50% of commercial antibodies fail to meet basic standards for characterization, potentially resulting in misleading research findings . Therefore, careful validation is essential to ensure that the detected signal genuinely represents PI-9 and not cross-reactivity with other proteins.

Additionally, researchers should consider using multiple antibodies targeting different epitopes of PI-9 to confirm findings, particularly for novel or contentious results. This approach helps mitigate issues related to potential epitope masking or conformational changes that might affect antibody recognition.

What are the optimal conditions for using PI-9 antibody in Western blotting experiments?

For optimal results in Western blotting experiments using PI-9 antibody, researchers should consider several key parameters based on the available data. The antibody concentration should be optimized based on the sample type: 2 μg/mL has been successfully used for mouse placenta lysate and K562 cell lysate, while 3 μg/mL has been used for human serum and recombinant human PI-9 protein .

Sample preparation is critical for preserving the native state of PI-9. Standard lysis buffers containing protease inhibitors should be used to prevent degradation during sample processing. For the detection of PI-9 in Western blotting, researchers should look for a band at approximately 42 kDa, which is the predicted molecular weight of the protein .

For blocking and antibody dilution, researchers typically use 5% non-fat dry milk or BSA in TBST or PBST. Secondary antibody selection should be based on the host species of the primary antibody; for rabbit polyclonal PI-9 antibodies, HRP-linked anti-rabbit antibodies (such as Guinea pig Anti-Rabbit at 1/2000 dilution) have been used successfully .

It's important to note that optimization may be required for specific experimental conditions and sample types. Researchers should perform initial titration experiments to determine the optimal antibody concentration that provides the best signal-to-noise ratio for their specific application.

How can PI-9 antibody be used to investigate the role of PI-9 in immune evasion by cancer cells?

PI-9 has been implicated in cancer immune evasion mechanisms, making it an important target for cancer immunology research. To investigate this role, researchers can employ PI-9 antibodies in multiple experimental approaches.

Immunohistochemical analysis of cancer tissues can reveal patterns of PI-9 expression across different tumor types and stages. The search results show that PI-9 antibody has been successfully used to stain bile duct cancer tissue and prostate cancer tissue , suggesting its utility in studying PI-9 expression in malignancies. Researchers can compare PI-9 expression levels between tumor tissue and adjacent normal tissue to identify potential correlations with disease progression or patient outcomes.

For mechanistic studies, researchers can use PI-9 antibody in co-culture experiments with immune effector cells (such as cytotoxic T lymphocytes or natural killer cells) and cancer cells. Immunofluorescence microscopy using PI-9 antibody can visualize PI-9 localization within cancer cells during immune cell interactions. This approach can help determine whether PI-9 expression levels correlate with resistance to immune cell-mediated killing.

Additionally, flow cytometry using PI-9 antibody can quantify PI-9 expression levels across cancer cell populations and correlate expression with other markers of immune evasion. For in vivo studies, researchers can use PI-9 antibody for immunohistochemical analysis of tumor samples from animal models, potentially correlating PI-9 expression with tumor growth rates or response to immunotherapy.

What are common technical challenges when using PI-9 antibody in IHC-P and how can they be addressed?

When using PI-9 antibody for immunohistochemistry on paraffin-embedded tissues (IHC-P), researchers may encounter several technical challenges that require careful optimization.

Antigen retrieval is often a critical step for successful IHC-P with PI-9 antibody. Formalin fixation can mask epitopes through protein cross-linking, potentially preventing antibody recognition of PI-9. Researchers should optimize antigen retrieval methods, testing both heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) and enzymatic methods, to determine which approach best exposes PI-9 epitopes while preserving tissue morphology.

Background staining can be another common issue in IHC-P. To minimize non-specific binding, researchers should optimize blocking conditions using bovine serum albumin (BSA), normal serum from the same species as the secondary antibody, or commercial blocking reagents. Thorough washing steps between incubations are also essential for reducing background.

Antibody concentration requires careful titration for IHC-P applications. While the search results indicate that 10 μg/ml has been used successfully for various tissue types , researchers should perform titration experiments to determine the optimal concentration for their specific tissue samples and experimental conditions.

How does antibody selection affect reproducibility in PI-9 research?

Antibody selection is a critical factor affecting research reproducibility, and this is particularly relevant for PI-9 studies. As highlighted in the literature, approximately 50% of commercial antibodies fail to meet basic standards for characterization, potentially resulting in financial losses of $0.4–1.8 billion per year in the United States alone due to irreproducible research .

When selecting PI-9 antibodies, researchers should prioritize antibodies that have been extensively characterized through multiple applications and validated in relevant biological systems. For example, antibodies that have been tested in Western blot, IHC-P, and ICC/IF provide greater confidence that they recognize PI-9 specifically across different experimental conditions .

Polyclonal versus monoclonal antibody selection presents different considerations. Polyclonal antibodies, like the rabbit polyclonal PI-9 antibody described in the search results , recognize multiple epitopes on the target protein, potentially providing higher sensitivity but with possible increased risk of cross-reactivity. Monoclonal antibodies recognize a single epitope, offering higher specificity but potentially lower sensitivity.

To enhance reproducibility, researchers should:

• Document detailed information about the antibody used, including vendor, catalog number, lot number, and dilution

• Include appropriate positive and negative controls in all experiments

• Validate antibody performance in their specific experimental system

• Consider using alternative antibodies or complementary techniques to confirm key findings

Recent initiatives like NeuroMab, which employs rigorous screening of approximately 1,000 clones across multiple assays , highlight the importance of thorough antibody characterization for research reproducibility.

What considerations are important when using PI-9 antibody for detecting protein-protein interactions?

When using PI-9 antibody to study protein-protein interactions, researchers should carefully consider several methodological aspects to obtain reliable and biologically meaningful results.

Epitope accessibility is a primary concern. If the PI-9 antibody's epitope overlaps with or is proximal to binding sites for interacting proteins (such as granzyme B), antibody binding might disrupt or prevent the protein-protein interaction being studied. Alternatively, protein binding partners might mask the antibody epitope, leading to false negative results. Researchers should consider using multiple antibodies targeting different regions of PI-9 to address this concern.

For co-immunoprecipitation (co-IP) experiments, preserving native protein structure and interactions is crucial. Lysis conditions should be mild enough to maintain protein-protein interactions while still effectively solubilizing PI-9. Non-ionic detergents such as NP-40 or Triton X-100 at low concentrations (0.1-1%) are often suitable. Researchers should also consider adding appropriate protease inhibitors to prevent degradation during the procedure.

When performing immunofluorescence studies to visualize co-localization, proper fixation is essential. Paraformaldehyde fixation (typically 4%) preserves protein localization while maintaining antigenicity for most antibodies. The data indicates that PI-9 antibody has been successfully used in immunocytochemistry/immunofluorescence with HeLa cells , suggesting its suitability for co-localization studies.

For proximity ligation assays (PLA) or FRET (Förster resonance energy transfer) analyses, which provide higher resolution information about protein-protein interactions, careful antibody pairing is essential. Researchers should select antibodies raised in different host species to facilitate simultaneous detection or use directly labeled primary antibodies to avoid potential steric hindrance from secondary antibodies.

How can PI-9 antibody be used in conjunction with other methodologies for comprehensive studies?

Integrating PI-9 antibody-based techniques with complementary methodologies creates powerful research approaches for understanding PI-9 biology in greater depth. These integrated approaches can provide more comprehensive insights than any single method alone.

Combining antibody-based detection with transcriptomic analyses allows researchers to correlate PI-9 protein expression with mRNA levels, providing insights into post-transcriptional regulation. For example, researchers could use PI-9 antibody for immunohistochemistry or Western blotting in conjunction with RT-qPCR or RNA-seq to determine whether protein and mRNA levels are concordant across different tissues or experimental conditions.

Mass spectrometry-based proteomics complements antibody-based detection by providing unbiased, quantitative information about PI-9 and its interacting partners. Immunoprecipitation using PI-9 antibody followed by mass spectrometry (IP-MS) can identify novel protein interactions, while targeted mass spectrometry can validate antibody-based quantification.

Functional assays measuring granzyme B activity can be correlated with PI-9 levels detected by antibody-based methods to establish structure-function relationships. For instance, researchers could use the PI-9 antibody to quantify protein levels across cell lines with varying sensitivity to granzyme B-mediated apoptosis, potentially revealing correlations between PI-9 expression and functional outcomes.

For tracking dynamic changes in PI-9 expression, researchers can combine live cell imaging techniques with fixed cell antibody staining. This approach could involve monitoring cells during immune interactions using reporter systems, followed by fixation and PI-9 antibody staining to correlate dynamic behaviors with PI-9 expression patterns.

What are the considerations for using PI-9 antibody in multiplex immunofluorescence studies?

Antibody compatibility is a primary consideration. When selecting antibodies for multiplexing with PI-9 antibody, researchers should choose primary antibodies raised in different host species to avoid cross-reactivity between secondary antibodies. Based on the search results, the rabbit polyclonal PI-9 antibody would need to be paired with antibodies raised in species other than rabbit, such as mouse, goat, or chicken.

Spectral overlap between fluorophores must be minimized to avoid bleed-through artifacts. Researchers should select fluorophores with well-separated excitation and emission spectra and perform appropriate controls, including single-color controls, to verify signal specificity. The search results indicate that PI-9 antibody has been used with FITC-linked secondary antibodies in immunofluorescence applications , which should be considered when designing a multiplex panel.

Sequential staining protocols may be necessary for more complex multiplexing. This approach involves applying and detecting one primary-secondary antibody pair, followed by elution or inactivation of these antibodies before applying the next set. This technique is particularly valuable when host species limitations prevent simultaneous staining.

For tissues with high autofluorescence, such as those containing lipofuscin or collagen, additional steps may be necessary to quench autofluorescence. Treatments with Sudan Black B or commercial autofluorescence quenchers should be tested to determine compatibility with PI-9 antibody staining.

How might advances in antibody engineering impact future research applications of PI-9 antibody?

Advances in antibody engineering are transforming the landscape of research antibodies, with significant implications for PI-9 research. These technological developments promise to address many current limitations while opening new experimental possibilities.

Recombinant antibody technology represents a major advance over traditional hybridoma-produced antibodies. Recombinant PI-9 antibodies would offer superior batch-to-batch consistency, eliminating the variability that can compromise experimental reproducibility. The genetic sequence information for recombinant antibodies is preserved, allowing for standardization across researchers and laboratories. This aligns with recent initiatives like NeuroMab, which has begun generating recombinant antibodies optimized for neuroscience research .

Site-specific conjugation technologies allow precise attachment of labels or functional groups to defined positions on antibodies, improving performance in applications like imaging or affinity purification. For PI-9 research, site-specifically labeled antibodies could provide enhanced sensitivity for detecting low-abundance expression while maintaining low background.

Smaller antibody formats, such as single-chain variable fragments (scFvs) or nanobodies derived from camelid antibodies, offer advantages for certain applications. Their reduced size compared to conventional antibodies enables better tissue penetration, potentially improving staining in thick tissue sections or whole organs. These smaller formats might also reduce the steric hindrance that can limit epitope accessibility in complex samples.

Bispecific antibodies, engineered to recognize two different epitopes simultaneously, could provide new tools for studying PI-9 in relation to its binding partners like granzyme B. Such antibodies could facilitate the study of complex formation or enable novel functional assays that require simultaneous recognition of multiple targets.

What emerging trends will shape the future of PI-9 antibody applications in research?

The field of PI-9 antibody research is poised for significant evolution, driven by advances in antibody technology, increased focus on reproducibility, and integration with emerging methodologies. Several key trends are likely to shape future applications.

The movement toward recombinant antibodies represents a fundamental shift in research antibody development. As highlighted in the antibody characterization literature, problems with variable quality of commercial antibodies have resulted in significant financial losses and irreproducible research . The transition to recombinant PI-9 antibodies with defined sequences will address these issues by providing consistent reagents with batch-to-batch reproducibility.

Increased emphasis on comprehensive validation standards is another important trend. Future PI-9 antibody research will likely adopt more rigorous validation approaches similar to those employed by initiatives like NeuroMab, which screens approximately 1,000 clones across multiple assays to ensure antibody performance in relevant applications . This trend aligns with broader efforts to enhance research reproducibility across biomedical sciences.

Integration of antibody-based methods with advanced imaging technologies such as super-resolution microscopy, expansion microscopy, and intravital imaging will provide unprecedented insights into PI-9 localization and dynamics at subcellular resolution and in living systems. These approaches will move beyond static snapshots of PI-9 expression to reveal its dynamics in physiological contexts.

The application of artificial intelligence and machine learning to image analysis will enhance the quantitative aspects of PI-9 antibody-based research. These computational approaches will enable more objective and comprehensive analysis of immunohistochemistry and immunofluorescence data, facilitating detection of subtle patterns that might be missed by conventional analysis.