PITPNA Antibody

What is PITPNA and why is it important in cellular research?

PITPNA (phosphatidylinositol transfer protein alpha) is a critical protein involved in lipid metabolism and intracellular signaling pathways. It plays an essential role in stimulating phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network, which promotes processes like insulin granule maturation . PITPNA is crucial for the transport of phosphatidylinositol between membrane compartments and participates in regulating key cellular processes including signal transduction and vesicular trafficking . Research interest in PITPNA has increased substantially due to its associations with type 2 diabetes and various cancers, making reliable antibodies for its detection imperative for advancing understanding of these disease mechanisms .

How do I choose the most appropriate PITPNA antibody for my research application?

Selecting the appropriate PITPNA antibody depends on several experimental factors:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IF/ICC, IP, or ELISA). For instance, antibody 16613-1-AP from Proteintech has been validated for WB, IP, IF, IHC, and ELISA applications , while CAB12966 has been validated for WB, IF/ICC, and ELISA applications .

• Species reactivity: Confirm the antibody reacts with your species of interest. Many PITPNA antibodies show reactivity with human, mouse, and rat samples .

• Epitope recognition: Consider which region of PITPNA your antibody targets. For example, CAB12966 targets a C-terminal sequence corresponding to amino acids 243-270 , while others may target different regions.

• Validation evidence: Review the validation data provided by manufacturers, including Western blot images, immunohistochemistry results, and published references using the antibody .

• Clonality choice: Determine whether a monoclonal or polyclonal antibody better suits your needs. Most available PITPNA antibodies are polyclonal (such as DF9746, A11279, and CAB12966) .

What are the recommended storage conditions for maintaining PITPNA antibody activity?

For optimal preservation of PITPNA antibody activity:

• Store at -20°C for long-term storage (typically stable for one year after shipment)

• For frequent use or short-term storage, keep at 4°C for up to one month

• Avoid repeated freeze-thaw cycles which can degrade antibody quality and reduce specificity

• Many PITPNA antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability

• For the 20μl size of some PITPNA antibodies, note that they may contain 0.1% BSA

• Aliquoting is recommended for antibodies that will be used multiple times to minimize freeze-thaw cycles, though some manufacturers note it is unnecessary for -20°C storage

How should I optimize PITPNA antibody dilutions for different experimental applications?

Optimization of PITPNA antibody dilutions requires methodical titration based on application type:

For Western Blot (WB):

• Begin with the manufacturer's recommended dilution range (e.g., 1:500-1:3000 for 16613-1-AP or 1:500-1:2000 for CAB12966 )

• Perform a dilution series experiment starting at the higher concentration (lower dilution) and increasing the dilution

• Include positive controls such as HEK-293 cells or human brain tissue, which have been validated to express detectable PITPNA levels

• Evaluate signal-to-noise ratio at each dilution, selecting the highest dilution that provides a clear specific band at approximately 32 kDa (the observed molecular weight of PITPNA)

For Immunohistochemistry (IHC):

• Start with the recommended dilutions (e.g., 1:20-1:200 for 16613-1-AP or 1:25 for some antibodies )

• For paraffin-embedded sections, test both antigen retrieval methods: TE buffer pH 9.0 and citrate buffer pH 6.0 to determine optimal conditions

• Validate using positive control tissues such as human lung, brain, or heart tissues

• Systematically increase dilutions until background is minimized while maintaining specific signal

For Immunoprecipitation (IP):

• Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

• Verify results using HEK-293 cells as a positive control

For Immunofluorescence (IF)/Immunocytochemistry (ICC):

• Begin with dilutions of 1:50-1:200

• Perform antibody incubation steps in a humidified chamber protected from direct light (primary antibody: 3-4 hours; secondary antibody: 1 hour)

What are the most common causes of non-specific binding when using PITPNA antibodies, and how can I minimize them?

Non-specific binding with PITPNA antibodies can arise from several factors:

• Suboptimal blocking:

  • Solution: Use 3-5% BSA or 5% non-fat dry milk in TBS-T for Western blot applications

  • For IHC/IF, ensure adequate blocking (typically 1 hour at room temperature with appropriate blocking buffer)

• Excessive antibody concentration:

  • Solution: Titrate the antibody as described above to find the minimum effective concentration

  • Follow manufacturer recommendations (e.g., WB: 1:500-1:3000, IHC: 1:20-1:200)

• Insufficient washing:

  • Solution: Increase the number and duration of wash steps (e.g., 3-5 washes of 5-10 minutes each with TBS-T)

  • Use gentle agitation during washing to improve removal of unbound antibody

• Cross-reactivity with similar epitopes:

  • Solution: Pre-absorb the antibody with the immunogen or use more specific PITPNA antibodies

  • Consider using monoclonal antibodies if polyclonal antibodies show cross-reactivity

• Sample preparation issues:

  • Solution: Ensure proper fixation and antigen retrieval methods

  • For IHC, test both TE buffer pH 9.0 and citrate buffer pH 6.0 as suggested for PITPNA antibodies

• Secondary antibody issues:

  • Solution: Include controls without primary antibody to assess secondary antibody specificity

  • Ensure secondary antibody is appropriate for the host species of the primary antibody (typically rabbit for PITPNA antibodies)

How do I accurately interpret PITPNA antibody results in the context of PITPNA-AS1 research?

Interpreting PITPNA antibody results in PITPNA-AS1 research requires careful consideration:

• Distinguish between protein and RNA detection:

  • PITPNA antibodies detect the protein itself, while PITPNA-AS1 is a long non-coding RNA that requires RNA detection methods

  • Do not use antibodies for direct detection of PITPNA-AS1; instead use RT-PCR, RNA-seq, or RNA FISH

• Understand the regulatory relationship:

  • PITPNA-AS1 may regulate PITPNA expression, so correlate protein levels (antibody detection) with PITPNA-AS1 RNA levels

  • In studies where PITPNA-AS1 acts as a competing endogenous RNA (ceRNA) to sponge miRNAs like miR-363-5p or miR-98-5p, PITPNA protein levels may be indirectly affected

• Disease-specific contexts:

  • In gastric cancer research, PITPNA-AS1 has been shown to be overexpressed and associated with poor survival

  • In hepatocellular carcinoma, PITPNA-AS1 functions as a ceRNA for miR-363-5p, regulating platelet-derived growth factor-D (PDGFD)

  • When studying such disease models, correlate PITPNA protein levels with PITPNA-AS1 expression to understand potential regulatory mechanisms

• Experimental controls:

  • Include cells/tissues with known PITPNA-AS1 knockdown or overexpression to understand how this affects PITPNA protein levels

  • Use appropriate controls for antibody specificity, especially when studying systems where PITPNA expression may be altered due to PITPNA-AS1 manipulation

• Cisplatin resistance models:

  • In studies of cisplatin resistance in gastric cancer, note that PITPNA-AS1 expression can be suppressed by cisplatin treatment

  • Consider how treatment conditions might affect both PITPNA-AS1 and PITPNA levels when interpreting antibody results

How should I approach PITPNA antibody use in pancreatic beta-cell and diabetes research?

When using PITPNA antibodies in diabetes research:

• Sample selection and preparation:

  • Human islets from T2D donors and non-diabetic controls are critical for comparative studies

  • When using mouse models, the conditional deletion of Pitpna in beta-cells (Ins-Cre, Pitpna mice) provides a relevant model

  • Proper preservation of islet architecture is crucial; use appropriate fixation protocols optimized for pancreatic tissue

• Methodological considerations:

  • For Western blot analysis of PITPNA in islet samples, ensure consistent loading by normalizing to housekeeping proteins

  • For immunohistochemistry, co-stain with insulin to specifically identify beta-cells and confirm PITPNA localization

  • In studies examining PITPNA restoration in T2D islets, carefully document baseline expression before intervention

• Data interpretation in diabetes context:

  • PITPNA expression is markedly reduced in beta-cells of T2D human subjects, with no change observed in alpha and gamma-cell populations

  • Unlike PITPNA, expression of the PITPNB isoform remains unchanged in beta-cells of T2D human donors compared to non-diabetic donors

  • PITPNA reduction correlates with decreased AGO2 levels in islets of T2D donors, which may reflect reduced capacity for compensatory proliferation

• Functional correlations:

  • When analyzing PITPNA expression, correlate with functional parameters such as glucose-stimulated insulin secretion (GSIS)

  • Examine insulin granule morphology and docking via transmission electron microscopy to relate PITPNA levels to granule formation

  • Consider analyzing proinsulin processing efficiency as PITPNA silencing leads to impaired insulin granule maturation

What special considerations apply when using PITPNA antibodies in cancer research?

For cancer research applications of PITPNA antibodies:

• Context of PITPNA and PITPNA-AS1 expression:

  • PITPNA-AS1 is overexpressed in various cancers including gastric cancer, hepatocellular carcinoma, and cervical cancer

  • When studying PITPNA protein expression in these cancers, consider the potential regulatory effect of PITPNA-AS1

• Cisplatin resistance studies:

  • PITPNA-AS1 expression is decreased in cisplatin-resistant gastric cancer tissues

  • Cisplatin treatment (at IC50 concentrations) can significantly suppress PITPNA-AS1 expression in gastric cancer cell lines like MKN45 and AGS

  • The mechanism for this downregulation may involve H3K27ac, a marker for active enhancers and promoters

• miRNA interactions:

  • PITPNA-AS1 acts as a ceRNA for miRNAs like miR-98-5p and miR-363-5p

  • When studying PITPNA protein levels in cancer, consider analyzing relevant miRNAs to understand potential post-transcriptional regulation

• Clinical correlations:

  • High PITPNA-AS1 expression correlates with poor survival in gastric cancer patients

  • PITPNA-AS1 expression positively associates with macrovascular invasion and advanced tumor stages in hepatocellular carcinoma

  • When examining PITPNA protein expression using antibodies, correlate findings with these clinical parameters

• Cell line selection:

  • For Western blot validation, use appropriate cell lines where PITPNA expression has been confirmed, such as HEK-293 cells

  • For cancer-specific studies, consider using cancer cell lines like MKN45 and AGS (gastric cancer), Huh7 and Hep3B (hepatocellular carcinoma)

What are the optimal protocols for using PITPNA antibodies in Western blot applications?

Detailed Western Blot Protocol for PITPNA Detection:

• Sample Preparation:

  • Extract proteins from tissues or cells using RIPA buffer with protease inhibitors

  • For brain tissue and HEK-293 cells (positive controls for PITPNA), ensure gentle homogenization to preserve protein integrity

  • Quantify protein concentration using Bradford or BCA assay

  • Prepare 20-50 μg of protein per lane with 4× Laemmli sample buffer and heat at 95°C for 5 minutes

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels (PITPNA has an observed molecular weight of 32 kDa)

  • Run gel at 80V through stacking gel, then increase to 120V through resolving gel

  • Include molecular weight markers to verify the correct band size

• Transfer:

  • Transfer proteins to PVDF or nitrocellulose membrane at 100V for 60-90 minutes in cold transfer buffer

  • Verify transfer efficiency with Ponceau S staining

• Blocking:

  • Block membrane with 5% non-fat dry milk or 3-5% BSA in TBS-T for 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute PITPNA antibody according to manufacturer's recommendations:

    • 16613-1-AP: 1:500-1:3000 dilution

    • DF9746: WB dilution not specified in the search results

    • A11279: 1:500-1:2000 dilution

    • CAB12966: 1:2000 dilution

  • Incubate membrane with diluted antibody in blocking buffer overnight at 4°C with gentle rocking

• Washing:

  • Wash membrane 3-5 times with TBS-T, 5-10 minutes each wash with gentle agitation

• Secondary Antibody Incubation:

  • Use HRP-conjugated anti-rabbit IgG secondary antibody (as most PITPNA antibodies are rabbit-derived)

  • Dilute according to manufacturer's recommendations (typically 1:5000-1:10000)

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using ECL substrate

  • Expose to X-ray film or use digital imaging system

  • Expected band at 32 kDa for PITPNA

• Validation:

  • Use positive controls such as HEK-293 cells or human brain tissue

  • Consider mouse brain, heart, kidney, or liver tissues as additional positive controls for rodent studies

What immunohistochemistry protocol modifications are necessary for optimal PITPNA detection in different tissues?

Optimized IHC Protocol for PITPNA Detection:

• Tissue Preparation:

  • For formalin-fixed paraffin-embedded (FFPE) sections, cut at 4-6 μm thickness

  • For frozen sections, cut at 8-10 μm thickness and fix appropriately

  • Use positive control tissues such as human lung, brain, or heart tissue

• Deparaffinization and Rehydration (for FFPE sections):

  • Deparaffinize in xylene (2 × 10 minutes)

  • Rehydrate through graded alcohols (100%, 95%, 80%, 70%) to water

• Antigen Retrieval (critical for PITPNA detection):

  • Primary recommendation: Use TE buffer pH 9.0 for heat-induced epitope retrieval

  • Alternative method: Use citrate buffer pH 6.0 if TE buffer yields suboptimal results

  • Perform heat-induced epitope retrieval using a pressure cooker, microwave, or water bath (95-99°C for 15-20 minutes)

  • Cool slides to room temperature (approximately 20 minutes)

• Peroxidase and Protein Blocking:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol for 10 minutes

  • Wash in PBS or TBS (3 × 5 minutes)

  • Block non-specific binding with 5-10% normal serum (from the same species as the secondary antibody) in PBS/TBS with 1% BSA for 30-60 minutes

• Primary Antibody Incubation:

  • Dilute PITPNA antibody according to recommendations:

    • 16613-1-AP: 1:20-1:200 dilution for IHC

    • Other antibodies: Follow manufacturer's recommendations (e.g., 1:25 for some PITPNA antibodies)

  • Incubate in a humidified chamber for 3-4 hours at room temperature or overnight at 4°C

  • Protect from direct light during incubation

• Washing:

  • Wash thoroughly in PBS or TBS (3 × 5 minutes)

• Secondary Antibody Incubation:

  • Apply appropriate biotinylated secondary antibody

  • Incubate for 1 hour at room temperature in a humidified chamber protected from direct light

• Detection and Visualization:

  • Use avidin-biotin complex (ABC) or polymer detection system

  • Develop with DAB or other appropriate chromogen

  • Counterstain with hematoxylin

  • Dehydrate through graded alcohols, clear in xylene, and mount

• Tissue-Specific Modifications:

  • Brain tissue: Extend antigen retrieval time to 25-30 minutes due to dense tissue

  • Pancreatic tissue: For diabetes research, co-stain with insulin antibody to identify beta-cells and correlate with PITPNA expression

  • Liver tissue: Reduce background by extending blocking time to 60 minutes

  • Cancer tissues: Consider dual staining with proliferation markers to correlate PITPNA expression with cell proliferation

How can I effectively use PITPNA antibodies in co-immunoprecipitation studies to identify interaction partners?

Optimized Co-Immunoprecipitation Protocol for PITPNA:

• Lysate Preparation:

  • Use HEK-293 cells as a positive control system for PITPNA immunoprecipitation

  • Harvest cells and lyse in a gentle IP lysis buffer (e.g., 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol with protease and phosphatase inhibitors)

  • Incubate on ice for 30 minutes with occasional mixing

  • Clarify lysate by centrifugation at 14,000g for 10 minutes at 4°C

  • Determine protein concentration (typically 1.0-3.0 mg of total protein is recommended for PITPNA IP)

• Pre-clearing (reduces non-specific binding):

  • Incubate lysate with protein A/G beads and normal rabbit IgG for 1 hour at 4°C with rotation

  • Remove beads by centrifugation

• Antibody Binding:

  • Add PITPNA antibody to pre-cleared lysate at recommended amounts:

    • For 16613-1-AP: 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

    • For other antibodies: Follow manufacturer's recommendations

  • Incubate overnight at 4°C with gentle rotation

• Immunoprecipitation:

  • Add pre-washed protein A/G beads to the lysate-antibody mixture

  • Incubate for 2-4 hours at 4°C with gentle rotation

  • Collect beads by brief centrifugation (1000g for 1 minute)

  • Wash beads 4-5 times with cold lysis buffer

• Elution:

  • For direct analysis of PITPNA-associated proteins:

    • Add 2× Laemmli sample buffer and heat at 95°C for 5 minutes

    • Separate proteins by SDS-PAGE and analyze by Western blot or mass spectrometry

  • For native elution to preserve protein interactions:

    • Use gentle elution buffer (e.g., 0.1 M glycine pH 3.0)

    • Neutralize immediately with 1 M Tris pH 8.0

• Controls and Validation:

  • Include IP with normal rabbit IgG as a negative control

  • Include input sample (5-10% of the lysate used for IP)

  • Verify PITPNA pull-down by Western blot using a portion of the IP sample

  • For confirmation of novel interactions, consider reverse co-IP using antibodies against the identified partners

• Identification of Interaction Partners:

  • Known PITPNA interactions include phosphatidylinositol 4-OH kinase, which it stimulates to produce PtdIns-4-P in the trans-Golgi network

  • In insulin-producing cells, examine interactions with proteins involved in insulin granule maturation and trafficking

  • For novel interaction discovery, analyze co-IP samples by mass spectrometry

• Analysis of Functional Significance:

  • For identified partners, perform functional validation through additional approaches:

    • Proximity ligation assay to confirm interactions in situ

    • Mutational analysis to map interaction domains

    • Functional assays to determine the physiological relevance of the interaction

How can PITPNA antibodies be used in conjunction with advanced imaging techniques for subcellular localization studies?

Integrating PITPNA Antibodies with Advanced Imaging:

• Super-resolution Microscopy:

  • STED (Stimulated Emission Depletion) Microscopy:

    • Use fluorophore-conjugated secondary antibodies with optimal STED properties (e.g., STAR 580, STAR 635P)

    • For PITPNA localization in the trans-Golgi network, co-stain with Golgi markers

    • Resolution of ~50 nm allows detailed visualization of PITPNA in membrane subdomains

  • STORM/PALM Techniques:

    • Employ photo-switchable fluorophores coupled to anti-PITPNA antibodies

    • Use direct STORM for quantitative single-molecule localization of PITPNA

    • Particularly useful for studying PITPNA clustering at membrane interfaces

• Live-Cell Imaging Adaptations:

  • SNAP/CLIP-tag Fusion Systems:

    • Generate PITPNA-SNAP fusion constructs for pulse-chase labeling

    • Use in conjunction with fixed-cell antibody staining to validate construct behavior

    • Enables tracking of PITPNA dynamics during phospholipid transfer events

  • Split-GFP Complementation:

    • Combine with antibody validation to verify interaction partners identified in co-IP studies

    • Useful for confirming PITPNA interactions with PI 4-kinase in living cells

• Correlative Light and Electron Microscopy (CLEM):

  • Immunogold labeling of PITPNA for high-resolution TEM after fluorescence imaging

  • Particularly valuable for examining PITPNA localization at insulin granules in beta-cells

  • Protocol modification: use 10 nm gold-conjugated secondary antibodies after primary PITPNA antibody incubation

• Expansion Microscopy:

  • Physical expansion of specimens allows super-resolution imaging on conventional microscopes

  • Protocol adaptation for PITPNA:

    • Use high-dilution antibody incubations (1:500-1:1000)

    • Extend primary antibody incubation to overnight at 4°C

    • Verify epitope accessibility after expansion by comparing with conventional IF

• Multiplexed Imaging:

  • Cyclic Immunofluorescence (CycIF):

    • Sequential imaging with different antibody sets

    • Include PITPNA antibody in appropriate cycle based on host species compatibility

    • Allows correlation of PITPNA with numerous markers in the same tissue section

  • Mass Cytometry Imaging (IMC):

    • Label PITPNA antibodies with rare earth metals

    • Combine with metal-labeled antibodies against interacting partners

    • Quantify PITPNA levels in heterogeneous tissues like pancreatic islets

What approaches can be used to investigate the impact of post-translational modifications on PITPNA using specific antibodies?

Strategies for Studying PITPNA Post-translational Modifications:

• PTM-specific Antibody Development and Validation:

  • For phosphorylation studies, develop antibodies against known or predicted PITPNA phosphorylation sites

  • Validate specificity using phosphatase treatment of samples as negative controls

  • For other modifications (ubiquitination, acetylation, etc.), verify antibody specificity with corresponding enzymatic treatments

• Combined Immunoprecipitation and PTM Detection:

  • First IP PITPNA using validated antibodies like 16613-1-AP (0.5-4.0 μg for 1.0-3.0 mg of total protein)

  • Then probe the immunoprecipitated material with PTM-specific antibodies

  • This two-step approach allows enrichment of PITPNA before PTM detection

• Mass Spectrometry Integration:

  • Immunoprecipitate PITPNA from cells/tissues under study

  • Perform tryptic digestion and analyze by LC-MS/MS

  • Map identified modifications to functional domains of PITPNA

  • Validate key findings with targeted antibody-based approaches

• Functional Correlation of PTMs:

  • In diabetes research, examine how PTMs of PITPNA differ between normal and T2D islets

  • Compare PTM patterns after treatments that alter PITPNA function (e.g., glucose stimulation)

  • Use site-directed mutagenesis to create PTM-mimetic or PTM-deficient PITPNA variants for functional studies

• Temporal Dynamics of PTMs:

  • Apply time-course studies after cellular stimulation (e.g., glucose challenge in beta-cells)

  • Synchronize cells and analyze PITPNA PTMs throughout the cell cycle

  • Correlate PTM changes with functional outcomes like insulin granule formation

• PTM Crosstalk Analysis:

  • Investigate how different modifications on PITPNA influence each other

  • Use combinations of PTM-specific antibodies in sequential IPs

  • Develop multiplexed detection methods for simultaneous analysis of multiple PTMs

• In Situ Proximity Ligation Assay (PLA):

  • Combine PITPNA antibody with PTM-specific antibodies in PLA

  • Allows visualization and quantification of modified PITPNA in its native cellular context

  • Particularly useful for low-abundance modifications that may be lost during extraction

How can single-cell analysis techniques be combined with PITPNA antibodies for heterogeneity studies in disease models?

Integrating PITPNA Antibodies with Single-cell Technologies:

• Single-cell Western Blot (scWestern):

  • Adapt PITPNA antibodies (e.g., 16613-1-AP at 1:500 dilution) for microfluidic scWestern platforms

  • Enables protein-level quantification of PITPNA in individual cells

  • Compare with single-cell RNA-seq data to identify post-transcriptional regulation

• Mass Cytometry (CyTOF) Applications:

  • Metal-conjugate PITPNA antibodies for CyTOF analysis

  • Combine with markers for cell type identification and functional state

  • Particularly valuable for analyzing islet cell populations in diabetes research, where PITPNA expression is reduced in beta-cells of T2D donors

  • Protocol modification: Increase antibody concentration by ~2-fold compared to flow cytometry applications

• Imaging Mass Cytometry:

  • Perform spatial analysis of PITPNA expression at single-cell resolution in tissue sections

  • Correlate with disease markers and microenvironmental features

  • Especially relevant for studying PITPNA in heterogeneous tumor samples

• Spatial Transcriptomics Correlation:

  • Combine PITPNA immunofluorescence with spatial transcriptomics

  • Allows correlation of protein expression with transcriptional profiles in the same tissue region

  • Useful for understanding transcriptional regulation of PITPNA in disease contexts

• Single-cell RNA-seq with Protein (CITE-seq) Integration:

  • Adapt PITPNA antibodies for CITE-seq applications

  • Enables simultaneous measurement of PITPNA protein and transcriptome in the same cells

  • Particularly valuable for understanding PITPNA regulation in beta-cells, where expression differences have been observed in T2D

• Protocol considerations for pancreatic islet single-cell analysis:

  • Gentle dissociation protocols to maintain cell viability and protein integrity

  • Immediate fixation to preserve PITPNA localization and protein-protein interactions

  • Multiplexed staining to distinguish beta-cells (insulin+) from other islet cell types

  • Integration with functional data (e.g., calcium imaging) to correlate PITPNA levels with cellular function

How can PITPNA antibodies be used to assess therapeutic interventions targeting the PITPNA pathway in diabetes?

PITPNA Antibody Applications in Diabetes Intervention Assessment:

• Monitoring PITPNA Restoration Therapies:

  • Use Western blot with PITPNA antibodies to quantify protein levels before and after therapeutic intervention

  • Research has shown that restoration of PITPNA in islets of T2D human subjects reverses beta-cell defects

  • Protocol recommendation: Use 16613-1-AP antibody (1:500-1:3000) for consistent detection in human islet samples

• Assessing Interventions on Insulin Granule Maturation:

  • Combine PITPNA antibodies with TEM to evaluate insulin granule morphology

  • PITPNA restoration should correct defects in granule size, maturation, and docking seen in T2D beta-cells

  • Quantitative assessment of granule parameters (size, electron density, distance from membrane) provides objective endpoints

• Functional Correlation with GSIS:

  • Correlate PITPNA expression levels (detected by antibodies) with glucose-stimulated insulin secretion

  • Effective interventions should demonstrate parallel improvements in PITPNA levels and functional GSIS responses

  • This correlation provides mechanistic validation of the therapeutic approach

• Islet Cell Composition Analysis:

  • Use PITPNA antibodies in multiplexed immunofluorescence to examine:

    • Beta-cell specific restoration of PITPNA

    • Effects on other islet cell populations (alpha, delta cells)

    • Changes in beta-cell mass after treatment

• Biomarker Development:

  • Standardize PITPNA antibody-based assays for potential use as biomarkers

  • Correlate PITPNA levels with clinical parameters (HbA1c, insulin requirements)

  • Potential for development of prognostic indicators based on PITPNA expression patterns

• Monitoring Proinsulin Processing:

  • Since PITPNA plays a role in insulin granule maturation, measure the proinsulin/insulin ratio

  • Use appropriate antibodies to distinguish proinsulin from mature insulin

  • Effective PITPNA-targeting therapies should normalize this ratio in T2D islets

What are the considerations for using PITPNA antibodies in cancer research and potential therapeutic development?

PITPNA Antibody Applications in Cancer Research:

• Expression Profiling in Different Cancer Types:

  • Use IHC with PITPNA antibodies (e.g., 16613-1-AP at 1:20-1:200) to profile expression across cancer types

  • Consider multiplexed staining with cancer stem cell markers and proliferation markers

  • Studies have shown PITPNA-AS1 is overexpressed in gastric cancer, hepatocellular carcinoma, and cervical cancer

• Correlation with PITPNA-AS1 and miRNA Networks:

  • Combine PITPNA protein detection with analysis of PITPNA-AS1 and relevant miRNAs

  • PITPNA-AS1 acts as a ceRNA for miR-363-5p in hepatocellular carcinoma and miR-98-5p in gastric cancer

  • This correlation helps understand post-transcriptional regulation mechanisms

• Chemoresistance Studies:

  • Monitor PITPNA and PITPNA-AS1 expression changes in cisplatin-resistant cancer models

  • PITPNA-AS1 is decreased in cisplatin-resistant gastric cancer tissues

  • Cisplatin treatment suppresses PITPNA-AS1 expression, potentially through H3K27ac regulation

• Patient Stratification Markers:

  • Develop standardized scoring systems for PITPNA expression in tumors

  • Correlate with clinical parameters such as tumor stage, invasion status, and patient survival

  • High PITPNA-AS1 expression associates with poor survival in gastric cancer patients

• Therapeutic Target Validation:

  • Use PITPNA antibodies to validate target engagement in preclinical models

  • Monitor changes in downstream pathways after therapeutic intervention

  • Combine with functional assays to assess impact on cancer cell proliferation, invasion, and apoptosis

• Protocol Adaptations for Cancer Tissues:

  • For heterogeneous tumor samples, use laser capture microdissection before Western blot analysis

  • For IHC of cancer tissues, optimize antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Include appropriate positive controls such as specific cancer cell lines (MKN45, AGS for gastric cancer; Huh7, Hep3B for hepatocellular carcinoma)