Ptprs Antibody

What is PTPRS and why is it important in scientific research?

PTPRS (also known as R-PTP-sigma or PTPσ) is a member of the protein tyrosine phosphatase (PTP) family that dephosphorylates phosphotyrosyl residues in proteins that are phosphorylated by protein-tyrosine kinases (PTKs) . This transmembrane protein contains an extracellular region, a single transmembrane segment, and two tandem intracellular catalytic domains, representing a receptor-type PTP . PTPRS is particularly significant in research due to its role as an essential regulator of signal transduction pathways, playing important roles in regulating many cellular processes in conjunction with PTKs . Recent studies have demonstrated its specific expression in plasmacytoid dendritic cells (pDCs) and its function as an inhibitory receptor that prevents spontaneous interferon production and immune-mediated intestinal inflammation .

What are the structural characteristics of commercially available PTPRS antibodies?

Commercial PTPRS antibodies are available in both polyclonal and monoclonal formats, with polyclonal antibodies like the 29415-1-AP being developed in rabbits using PTPRS fusion protein immunogens . These antibodies typically target specific epitopes within the PTPRS protein structure and are supplied in liquid form with specific storage buffers (such as PBS with 0.02% sodium azide and 50% glycerol, pH 7.3) . The host species is commonly rabbit with IgG isotype, and purification is typically achieved through antigen affinity methods . The antibodies are designed to recognize PTPRS in various applications including Western Blot (WB), Immunohistochemistry (IHC), and ELISA, with demonstrated reactivity toward human and mouse samples .

How does the molecular weight of PTPRS observed in experiments compare with theoretical predictions?

There is a notable discrepancy between the calculated and observed molecular weights of PTPRS. While the calculated molecular weight based on amino acid sequence is approximately 217 kDa, the observed molecular weight in laboratory experiments is typically around 140 kDa . This discrepancy may be attributed to several factors including post-translational modifications, proteolytic processing, alternative splicing, or the effects of protein folding on electrophoretic mobility. When conducting Western blot analysis, researchers should expect to observe PTPRS at approximately 140 kDa rather than at its theoretical weight .

What are the recommended dilutions and protocols for using PTPRS antibodies in Western Blot applications?

For Western Blot applications using PTPRS antibodies such as 29415-1-AP, the recommended dilution range is 1:2000-1:12000 . The optimal dilution may vary depending on sample type and protein expression levels, so initial titration experiments are advised. Standard Western blot protocols should be followed, including proper sample preparation, SDS-PAGE separation, efficient transfer to membranes, and appropriate blocking steps. PTPRS antibodies have been validated for detection in various cell lines including HEK-293, A549, and HeLa cells . As with all antibodies, researchers should include appropriate positive and negative controls to validate specificity and performance.

What protocols should be followed for immunohistochemistry with PTPRS antibodies?

For immunohistochemistry (IHC) applications, PTPRS antibodies are typically used at dilutions ranging from 1:50 to 1:500 . The protocol requires careful antigen retrieval, with suggested methods including TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 . Positive IHC detection has been validated in mouse brain tissue . As with all IHC procedures, optimization of antigen retrieval conditions, antibody concentration, incubation times, and detection systems may be necessary for specific tissue types. Researchers should be aware that the efficacy of PTPRS antibodies in IHC may vary depending on tissue fixation methods and processing protocols.

How can PTPRS antibodies be used to study its function in plasmacytoid dendritic cells (pDCs)?

PTPRS antibodies can be utilized to investigate the functional role of PTPRS in pDCs through several methodological approaches:

• Surface staining and flow cytometry analysis to detect PTPRS expression specifically on pDCs in peripheral blood mononuclear cells (PBMCs)

• Antibody-mediated crosslinking experiments to assess the inhibitory effect on pDC activation. Research has demonstrated that such crosslinking inhibits pDC activation

• Combination with knockdown studies to compare effects of antibody binding versus reduced PTPRS expression. Previous studies showed that PTPRS knockdown enhanced interferon responses in pDC cell lines

• Comparative analysis between human and murine systems, as PTPRS is evolutionarily conserved and specifically expressed in pDCs in both species

• Co-expression studies with related proteins such as PTPRF, particularly in murine models where both are specifically expressed in pDCs

How do antibody-induced dimerization mechanisms affect PTPRS function and what implications does this have for experimental design?

Based on insights from related receptor tyrosine phosphatases like PTPRD, antibody-induced dimerization can significantly impact PTPRS function . When designing experiments with PTPRS antibodies, researchers should consider that binding to the extracellular domain may induce dimerization that inhibits phosphatase activity and potentially triggers proteolytic degradation through intracellular pathways . Different antibodies targeting different epitopes may have varying effects on dimerization and subsequent functional consequences. For example, the effect observed with monoclonal antibody 4C3, which interacts with the fibronectin domain of the extracellular portion and decreases phosphatase activity, differs from that of other antibodies . Experimental designs should include appropriate controls to account for these potential antibody-mediated effects that may confound the interpretation of results.

What is the significance of PTPRS in immune regulation and how can antibodies help elucidate these mechanisms?

PTPRS serves as an inhibitory receptor specifically on pDCs, playing a crucial role in preventing spontaneous interferon production and immune-mediated inflammation . Research utilizing PTPRS antibodies has revealed that PTPRS expression is inversely correlated with pDC activation, and antibody-mediated crosslinking inhibits cytokine production by these cells . Studies in mouse models have demonstrated that reduction of Ptprs (along with its homolog Ptprf) enhances interferon production by pDCs and causes mild intestinal inflammation . These findings position PTPRS antibodies as valuable tools for studying immune regulation in contexts such as:

• Autoimmune disorders where pDC hyperactivation may contribute to pathology

• Anti-viral immune responses where pDC-derived interferons are critical

• Cancer immunotherapy approaches targeting innate immune activation

• Inflammatory bowel diseases where intestinal immune homeostasis is disrupted

When designing such studies, researchers should consider both blocking and crosslinking effects of different antibodies, as these can produce opposite functional outcomes.

How can PTPRS antibodies be used to identify specific signaling pathways regulated by this phosphatase?

To identify specific signaling pathways regulated by PTPRS using antibodies, researchers can implement several methodological approaches:

• Immunoprecipitation followed by mass spectrometry to identify binding partners and potential substrates

• Phosphoproteomic analysis comparing cells before and after antibody-mediated PTPRS inhibition to identify hyperphosphorylated proteins

• Proximity labeling techniques combined with PTPRS antibodies to identify proteins in the vicinity of PTPRS in living cells

• Co-immunoprecipitation studies to validate specific protein-protein interactions

• Phospho-specific Western blotting focusing on candidate substrates to assess changes in phosphorylation status following PTPRS modulation by antibodies

When interpreting results, researchers should consider that antibody binding might induce artificial dimerization or crosslinking effects that may not reflect physiological regulation of PTPRS activity.

What controls should be included when using PTPRS antibodies to ensure experimental validity?

To ensure experimental validity when using PTPRS antibodies, several controls should be implemented:

• Positive control samples: Use cell lines with confirmed PTPRS expression such as HEK-293, A549, or HeLa cells for Western blot applications

• Negative control samples: Utilize PTPRS knockout cells or tissues, or cell lines known not to express PTPRS

• Competing peptide controls: Pre-incubation of the antibody with PTPRS extracellular domain fusion protein should block glomerular binding and abolish permeability activity

• Isotype control antibodies: Include irrelevant antibodies of the same isotype to control for non-specific binding

• Loading controls: For Western blot applications, include housekeeping proteins to normalize expression levels

• Secondary antibody-only controls: To detect non-specific binding of secondary antibodies

• Cross-reactivity controls: Test tissues from different species when assessing antibody specificity across species

These controls help distinguish specific from non-specific signals and validate experimental findings, particularly in complex applications like immunohistochemistry or flow cytometry.

How can researchers optimize antibody dilution for different experimental applications?

Optimization of PTPRS antibody dilution is crucial for obtaining reliable and reproducible results across different experimental applications. The recommended approach varies by application:

For each application, prepare a series of dilutions spanning the recommended range and evaluate signal intensity, background levels, and signal-to-noise ratio. The optimal dilution provides maximum specific signal with minimal background. Remember that optimal dilutions may vary depending on sample type, protein expression levels, and detection methods used . It is recommended that researchers titrate the antibody in each testing system to obtain optimal results .

How does PTPRS function differ between cell types and what implications does this have for antibody-based studies?

PTPRS demonstrates notable cell type-specific functions, particularly in neural tissues and immune cells like pDCs. In human immune cells, PTPRS expression is predominantly restricted to pDCs, with minimal expression in other leukocyte populations . This contrasts with its broader expression pattern in neural tissues. The function of PTPRS as an inhibitory receptor on pDCs represents an evolutionarily conserved mechanism to prevent spontaneous interferon production .

When designing antibody-based studies, researchers must consider:

• Cell type-appropriate positive and negative controls

• Potential cross-reactivity with homologous phosphatases in different cell types

• Variation in accessibility of antibody epitopes due to cell type-specific post-translational modifications

• Differences in signaling partners and downstream effectors between cell types

• Differential expression levels affecting antibody titration requirements

Understanding these cell type-specific differences is essential for correctly interpreting antibody-based studies and avoiding overgeneralization of findings from one cellular context to another.

What are the current challenges in developing highly specific antibodies against PTPRS and related phosphatases?

Developing highly specific antibodies against PTPRS presents several challenges:

• Sequence homology with related phosphatases (particularly PTPRD and PTPRF), which may lead to cross-reactivity

• Conservation across species, which can complicate the generation of antibodies recognizing species-specific epitopes

• Complex structure with multiple domains, limiting the accessibility of certain epitopes

• Various isoforms resulting from alternative splicing, which may not all be recognized by a single antibody

• Post-translational modifications that may mask epitopes or alter antibody binding efficiency

To address these challenges, researchers are employing strategies such as targeting unique regions within the extracellular domain, using recombinant protein fragments as immunogens, and extensive validation through knockout controls. Advanced antibody engineering techniques, including phage display and rational design approaches, are being explored to enhance specificity for PTPRS over related phosphatases.

How can PTPRS antibodies contribute to understanding the role of this phosphatase in disease mechanisms?

PTPRS antibodies represent valuable tools for investigating this phosphatase's role in various disease mechanisms. Research has already implicated PTPRS in several pathological processes:

• Immune dysregulation: Studies have shown that PTPRS deficiency combined with PTPRF deficiency leads to enhanced interferon response of pDCs, leukocyte infiltration in the intestine, and mild colitis . PTPRS antibodies can help investigate these mechanisms in inflammatory bowel diseases.

• Neurological disorders: As a member of the receptor tyrosine phosphatase family implicated in neuronal development and function, PTPRS may be involved in neurological conditions. Antibodies can help map its expression and activity in neural tissues in disease states.

• Cancer biology: Alterations in protein tyrosine phosphorylation are hallmarks of many cancers. PTPRS antibodies can be used to assess expression changes in tumor samples and investigate potential roles in tumor suppression or progression.

• Metabolic disorders: PTPs play roles in insulin signaling. PTPRS antibodies could contribute to understanding its potential involvement in metabolic regulation.

Methodologically, PTPRS antibodies can be employed in tissue microarrays for high-throughput screening across multiple disease samples, used for immunoprecipitation to identify disease-specific interaction partners, and applied in functional assays to assess how disease-associated mutations affect PTPRS activity and regulation.