inppl1a Antibody

What is INPPL1/SHIP-2 and why is it significant in research?

INPPL1 (Inositol Polyphosphate Phosphatase-Like 1), also known as SHIP-2 (SH2 domain-containing inositol 5'-phosphatase 2), is a critical enzyme involved in phosphoinositide signaling pathways. This 139 kDa protein regulates cellular processes including metabolism, proliferation, and cytoskeletal organization by catalyzing the dephosphorylation of phosphatidylinositol phosphates. INPPL1 contains several functional domains including an SH2 domain that mediates protein-protein interactions and a catalytic 5'-phosphatase domain. Research interest in INPPL1 has grown due to its implications in insulin signaling, diabetes, cancer progression, and other pathophysiological conditions. Antibodies against INPPL1 are essential tools for studying its expression, localization, interactions, and post-translational modifications in various experimental contexts .

What are the key applications for INPPL1/SHIP-2 antibodies in research?

INPPL1/SHIP-2 antibodies are versatile tools employed across multiple experimental techniques. Western blotting (WB) applications allow researchers to detect and quantify INPPL1 protein expression in cell and tissue lysates, typically revealing a band at approximately 139 kDa. Immunohistochemistry (IHC) using INPPL1 antibodies enables visualization of protein distribution in tissue sections, providing insights into tissue-specific expression patterns, as demonstrated in human heart and kidney tissues . Immunoprecipitation (IP) allows isolation of INPPL1 and its binding partners from complex biological samples, facilitating the study of protein-protein interactions. Immunofluorescence (IF) applications permit subcellular localization studies of INPPL1, revealing its distribution patterns within cells. Additionally, enzyme-linked immunosorbent assays (ELISA) using these antibodies enable quantitative detection of INPPL1 in various sample types .

How should I select the appropriate INPPL1/SHIP-2 antibody for my specific research needs?

Selecting the optimal INPPL1 antibody requires consideration of multiple experimental factors. First, determine your application requirements (WB, IHC, IP, IF) and verify that your antibody candidate has been validated for these specific applications. For example, ab166916 has been validated for IHC-P, WB, and ICC/IF applications with human, mouse, and rat samples . Second, consider species reactivity - ensure the antibody recognizes INPPL1 in your experimental species. Many INPPL1 antibodies react with human and mouse INPPL1, but verification for other species may be necessary .

Third, determine whether you need a monoclonal (higher specificity, consistent lot-to-lot performance) or polyclonal antibody (potentially higher sensitivity, recognition of multiple epitopes). For phosphorylation-specific studies, specialized antibodies targeting specific phosphorylation sites (e.g., pTyr1135) are available . Finally, review validation data provided by manufacturers, including Western blot images showing expected band sizes, knockout validation data, and immunohistochemistry images demonstrating specific staining patterns in relevant tissues . Cross-referencing with published literature using your antibody of interest can provide additional confidence in antibody performance and specificity.

What controls should I implement when working with INPPL1/SHIP-2 antibodies?

Implementing appropriate controls is essential for generating reliable and interpretable data with INPPL1 antibodies. Positive controls should include cell lines or tissues known to express INPPL1, such as HeLa or K562 cells, which have been documented to express detectable levels of the protein . Negative controls can include INPPL1 knockout cell lines, such as the INPPL1 knockout A549 cell line, which has been shown to lack the 120-150 kDa band corresponding to INPPL1 in Western blot analysis .

For immunoprecipitation experiments, include an isotype control IgG to establish baseline non-specific binding . When performing immunohistochemistry or immunofluorescence, include a primary antibody omission control and consider peptide competition assays to demonstrate staining specificity. For phospho-specific antibodies like those targeting pTyr1135, include controls with and without treatments that affect phosphorylation status . Loading controls in Western blots (e.g., GAPDH, β-actin, calnexin) are essential for normalizing protein levels across samples. When possible, validate findings using a second antibody recognizing a different epitope of INPPL1 to confirm specificity of your observations .

How can I optimize immunoprecipitation protocols using INPPL1/SHIP-2 antibodies?

Optimizing immunoprecipitation (IP) with INPPL1 antibodies requires careful attention to multiple experimental parameters. Begin with antibody selection - choose antibodies specifically validated for IP applications, such as ab70267, which has demonstrated successful IP of INPPL1 from HeLa cell lysates . The antibody amount is critical; typical starting concentrations range from 1-10 μg per IP reaction, with specific examples showing effective IP using 6 μg of ab70267 per reaction with 1 mg of total protein lysate .

The lysis buffer composition significantly impacts IP efficiency - use buffers containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease/phosphatase inhibitors. Pre-clearing lysates with protein A/G beads for 1 hour at 4°C helps reduce non-specific binding. For optimal antigen-antibody interaction, perform overnight incubation at 4°C with gentle rotation. After IP, implement stringent washing conditions (typically 3-5 washes) to remove non-specifically bound proteins while preserving specific interactions. For detection by Western blot, the same antibody can be used at a reduced concentration (e.g., 0.4 μg/mL as shown with ab70267) or a second antibody recognizing a different epitope for confirmation of specificity . For studying INPPL1-interacting proteins, consider crosslinking approaches or proximity-based labeling methods to capture transient interactions.

What approaches are recommended for studying INPPL1 phosphorylation sites and their functional significance?

Investigating INPPL1 phosphorylation requires specialized antibodies and methodological approaches. Phospho-specific antibodies targeting individual phosphorylation sites, such as the anti-INPPL1 (pTyr1135) antibody, enable direct detection of site-specific phosphorylation events . These antibodies can be employed in Western blotting to monitor changes in phosphorylation status following cellular treatments, stimuli, or genetic manipulations. When studying novel phosphorylation sites, validation through multiple approaches is essential, including phosphatase treatment controls and mutation of the targeted phosphorylation site to a non-phosphorylatable residue (e.g., Tyr to Phe).

Mass spectrometry-based phosphoproteomics provides comprehensive identification of multiple phosphorylation sites simultaneously. For functional studies, comparing wild-type INPPL1 with phospho-mimetic (e.g., Tyr to Glu) or phospho-deficient (e.g., Tyr to Phe) mutants can reveal the significance of specific phosphorylation events on INPPL1 enzymatic activity, protein-protein interactions, or subcellular localization. Kinase inhibitor screenings can help identify upstream regulators of INPPL1 phosphorylation. The functional relevance of phosphorylation at Tyr1135 specifically can be investigated using phospho-specific antibodies in combination with cellular assays measuring phosphoinositide metabolism, insulin signaling, or cytoskeletal reorganization . Consider temporal dynamics of phosphorylation events using time-course experiments with various stimuli relevant to INPPL1 function.

How can INPPL1 antibodies be utilized to investigate protein-protein interactions in signaling pathways?

INPPL1 antibodies serve as valuable tools for elucidating protein interaction networks within complex signaling cascades. Co-immunoprecipitation (Co-IP) represents a foundational approach, where INPPL1 antibodies can precipitate INPPL1 along with its interacting partners from cell lysates. For example, research has demonstrated interactions between INPPL1 and adapter proteins like APPL1, which connects INPPL1 to receptor signaling pathways . For these experiments, optimizing lysis conditions is critical - milder detergents (0.3-0.5% NP-40) help preserve protein-protein interactions while effectively solubilizing membrane-associated INPPL1 complexes.

Proximity ligation assays (PLA) using INPPL1 antibodies in combination with antibodies against suspected interaction partners allow visualization and quantification of protein-protein interactions in situ with high sensitivity. For analyzing dynamic interactions, live-cell imaging techniques including fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) combined with immunofluorescence validation using INPPL1 antibodies provide temporal and spatial resolution of interaction events. Additionally, pull-down assays using recombinant INPPL1 domains can identify direct binding partners, with subsequent validation using INPPL1 antibodies. The β-galactosidase assay described in search result 5 exemplifies how protein interaction studies can be conducted in heterologous systems, with similar approaches applicable using INPPL1 antibodies to validate interactions discovered through screening methods .

What are common challenges when using INPPL1/SHIP-2 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with INPPL1 antibodies. Background or non-specific staining in immunohistochemistry and immunofluorescence applications can be mitigated through optimized blocking (3-5% BSA or serum matching the secondary antibody host species), increased antibody dilution, and proper antigen retrieval methods (as specified for ab166916, which requires citrate buffer pH 6) . For Western blotting, multiple bands or unexpected molecular weights may occur; addressing this requires optimization of sample preparation (complete denaturation, fresh sample preparation), blocking conditions, and antibody concentration. The expected molecular weight for INPPL1 is 139 kDa, but post-translational modifications can result in observed sizes ranging from 120-150 kDa .

Weak or absent signal can result from insufficient protein expression, sample degradation, or suboptimal antibody concentration. To address this, increase protein loading (15-50 μg for Western blot as demonstrated with HeLa lysates) , optimize antibody concentration (starting with manufacturer recommendations, such as 1/100 dilution for IHC or 1/1000 for Western blot with ab166916) , and ensure proper sample handling to prevent protein degradation. For phospho-specific antibodies, such as those targeting pTyr1135, ensure samples are prepared with phosphatase inhibitors and appropriate stimulation conditions to maintain phosphorylation status . Cross-reactivity issues can be resolved through careful antibody selection, validation with knockout controls as demonstrated with INPPL1 knockout A549 cells , and peptide competition assays to confirm specificity.

How should I validate the specificity of INPPL1/SHIP-2 antibodies in my experimental system?

Rigorous validation of INPPL1 antibodies is essential for generating reliable research data. The gold standard approach involves comparing antibody reactivity between wild-type and INPPL1 knockout samples. As demonstrated with ab166916, testing in INPPL1 knockout A549 cell lysates alongside wild-type controls provides definitive evidence of antibody specificity - the expected 120-150 kDa band should be absent in knockout samples . When knockout models are unavailable, siRNA or shRNA knockdown of INPPL1 can serve as an alternative, expecting significantly reduced signal intensity proportional to knockdown efficiency.

Peptide competition assays offer another validation strategy, where pre-incubating the antibody with its immunizing peptide should abolish specific staining. For phospho-specific antibodies, such as those targeting pTyr1135, treatment with phosphatases should eliminate the signal . Cross-validation using multiple antibodies targeting different INPPL1 epitopes provides additional confidence when consistent results are obtained. Western blotting should reveal bands of expected molecular weight (approximately 139 kDa for INPPL1), while immunostaining patterns should be consistent with known INPPL1 biology and localization patterns. Comparing your results with published literature utilizing the same or different INPPL1 antibodies helps establish consensus on expression patterns and localization. Finally, mass spectrometry analysis of immunoprecipitated proteins can provide unbiased confirmation of antibody specificity.

How can INPPL1/SHIP-2 antibodies contribute to understanding its role in disease mechanisms?

INPPL1/SHIP-2 antibodies serve as critical tools for investigating disease mechanisms across multiple pathological conditions. In metabolic disease research, INPPL1 antibodies enable analysis of expression and phosphorylation changes in insulin-sensitive tissues from diabetes models, providing insights into insulin resistance mechanisms. Immunohistochemical analysis using antibodies like ab166916 permits visualization of INPPL1 distribution in tissues such as heart and kidney, which are significantly affected in cardiometabolic diseases . For cancer research, INPPL1 antibodies facilitate investigation of expression level alterations across different cancer types and can be employed in tissue microarrays to correlate expression with clinical outcomes.

Phospho-specific antibodies targeting sites such as pTyr1135 allow researchers to monitor how disease states and therapeutic interventions impact INPPL1 post-translational modifications and subsequent signaling pathway activation . In neurodegenerative disease models, where phosphoinositide signaling is frequently dysregulated, INPPL1 antibodies can reveal alterations in protein localization or expression. Co-immunoprecipitation with INPPL1 antibodies followed by mass spectrometry enables identification of disease-specific interaction partners. Additionally, these antibodies are valuable for validating potential therapeutic approaches targeting INPPL1, such as small molecule inhibitors or gene therapy strategies. In viral infection research, particularly relevant to HIV studies, antibodies to phosphoinositide pathways have demonstrated neutralizing capabilities, suggesting potential applications for INPPL1 antibodies in understanding host-pathogen interactions .

What methodological considerations are important when investigating INPPL1 in different tissue and cell types?

Investigating INPPL1 across diverse biological systems requires tailored methodological approaches. Tissue-specific extraction protocols significantly impact INPPL1 recovery and detection quality. For adipose and muscle tissues, which are particularly relevant for metabolism studies, specialized lysis buffers containing higher detergent concentrations (1-2% Triton X-100) and mechanical disruption methods improve protein extraction efficiency. When working with brain tissues, where lipid content is high, modified extraction protocols incorporating lipid solubilization agents may be necessary for complete INPPL1 recovery.

The choice of antibody should be guided by published validation data in your tissue or cell type of interest. For novel applications, preliminary validation steps including Western blotting to confirm expected molecular weight and knockdown/knockout controls are strongly recommended. Finally, for quantitative comparisons across tissue types, technical normalization approaches accounting for matrix effects and tissue-specific background should be implemented to ensure accurate interpretation of expression differences.

What innovative applications of INPPL1/SHIP-2 antibodies are emerging in current research?

Emerging research applications for INPPL1/SHIP-2 antibodies are expanding our understanding of phosphoinositide signaling networks. Single-cell Western blotting technologies combined with INPPL1 antibodies now enable quantification of expression heterogeneity within complex tissues, revealing previously undetectable subpopulations with distinct INPPL1 expression patterns. Super-resolution microscopy techniques (STORM, PALM, SIM) utilizing fluorophore-conjugated INPPL1 antibodies provide unprecedented visualization of subcellular localization and co-localization with binding partners at nanometer resolution, far beyond conventional microscopy capabilities.

Proximity labeling approaches (BioID, APEX) coupled with INPPL1 antibodies for validation are revolutionizing the mapping of dynamic INPPL1 interactomes under various cellular conditions. In the emerging field of spatial transcriptomics, INPPL1 antibodies are being integrated into multiplexed protein/RNA detection platforms to correlate protein expression with transcriptional programs across tissue microenvironments. Mass cytometry (CyTOF) applications incorporating metal-conjugated INPPL1 antibodies enable simultaneous measurement of INPPL1 with dozens of other proteins in single cells, providing comprehensive signaling network analysis.

For therapeutic development, INPPL1 antibodies are contributing to target engagement studies for small molecule inhibitors and facilitating screening of novel phosphoinositide pathway modulators. The development of intrabodies derived from INPPL1 antibodies presents opportunities for real-time monitoring of INPPL1 dynamics in living cells. Finally, humanized INPPL1 antibodies show potential in neutralizing pathogenic interactions, building upon observations with anti-phosphatidylinositol phosphate antibodies in viral neutralization contexts .

What experimental protocols are recommended for studying INPPL1 interactions with phospholipids?

Investigating INPPL1's interactions with phospholipids requires specialized methodologies that leverage antibodies as validation tools. Protein-lipid overlay assays (PLO) represent an accessible starting point, where purified phospholipids are spotted on membranes, incubated with recombinant INPPL1, and detected using INPPL1 antibodies. This approach allows identification of binding preferences across phosphoinositide species. For more quantitative binding measurements, surface plasmon resonance (SPR) or biolayer interferometry (BLI) with immobilized phospholipids and recombinant INPPL1, followed by antibody validation, provides binding kinetics data.

Liposome flotation assays offer another powerful approach - liposomes containing specific phospholipids are incubated with INPPL1, subjected to density gradient centrifugation, and fractions are analyzed by Western blotting with INPPL1 antibodies to determine phospholipid binding specificity. For cellular contexts, FRET-based biosensors can be employed alongside immunofluorescence validation with INPPL1 antibodies to monitor dynamic lipid-protein interactions in living cells. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled with antibody-based validation helps identify specific INPPL1 domains involved in phospholipid binding.

When studying INPPL1's phosphatase activity toward specific phospholipid substrates, in vitro phosphatase assays using purified recombinant INPPL1 (immunoprecipitated or commercially available) with defined phosphoinositide substrates allow quantification of enzymatic activity. Cellular studies can employ metabolic labeling of phosphoinositides followed by immunoprecipitation with INPPL1 antibodies to analyze associated lipids. Finally, lipid mass spectrometry analysis of samples from INPPL1 knockout or overexpression systems, validated with antibodies, provides comprehensive assessment of INPPL1's impact on cellular phosphoinositide profiles.

How should I design experiments to address contradictory results with INPPL1/SHIP-2 antibodies?

When confronted with contradictory results using INPPL1 antibodies, systematic troubleshooting and experimental design are essential. First, perform a comprehensive antibody validation comparison using multiple INPPL1 antibodies targeting different epitopes (such as ab70267 and ab166916) in parallel experiments under identical conditions . Validation should include Western blot analysis to confirm expected molecular weight (139 kDa), immunostaining pattern comparison, and ideally, testing in INPPL1 knockout or knockdown systems as demonstrated with the INPPL1 knockout A549 cell line .

Design carefully controlled experiments addressing potential confounding factors. For contradictory expression data, standardize sample preparation methods, protein quantification techniques, and loading controls. Implement both technical replicates (same sample, multiple measurements) and biological replicates (independent samples) with appropriate statistical analysis. For inconsistent localization data, systematically test fixation conditions, permeabilization methods, and antibody concentrations. Consider that INPPL1 localization may genuinely vary with cell type, cell cycle stage, or treatment conditions - time course experiments and co-localization with compartment markers can clarify these dynamics.

When contradictions exist between antibody-based results and other techniques (e.g., RNA-seq data showing expression while protein is undetectable), investigate regulatory mechanisms including post-transcriptional regulation, protein stability, or context-dependent expression. Consider orthogonal approaches such as CRISPR tagging of endogenous INPPL1 with fluorescent proteins or epitope tags to validate antibody findings. The integration of multiple methodologies (e.g., combining immunoblotting, immunofluorescence, and functional assays) provides stronger evidence than any single approach and helps resolve apparent contradictions.

What quantitative analysis methods are appropriate for INPPL1/SHIP-2 antibody-based experiments?

Quantitative analysis of INPPL1 antibody-based experiments requires rigorous methodological approaches. For Western blot densitometry, use digital image analysis software with linear dynamic range detection to quantify band intensities. Normalize INPPL1 signals to appropriate loading controls (GAPDH, β-actin, or calnexin as shown in some experiments with ab166916) and report results as relative expression rather than absolute values . For phospho-specific antibody experiments, such as those using anti-pTyr1135 antibodies, calculate the ratio of phosphorylated to total INPPL1 using parallel blots or sequential probing with phospho-specific and total INPPL1 antibodies .

In immunofluorescence quantification, establish standardized image acquisition parameters (exposure time, gain settings) across all samples and experimental conditions. Perform analysis on raw, unprocessed images, measuring mean fluorescence intensity within defined regions of interest or using automated cell segmentation algorithms. For colocalization analysis, employ quantitative metrics such as Pearson's correlation coefficient or Manders' overlap coefficient rather than subjective visual assessment.

For high-throughput applications such as tissue microarrays or multi-well plate formats, implement automated image analysis pipelines with consistent thresholding algorithms. Statistical analysis should employ appropriate tests based on data distribution (parametric or non-parametric) and experimental design (paired or unpaired comparisons). When comparing multiple experimental groups, use ANOVA with appropriate post-hoc tests and corrections for multiple comparisons. For all quantitative analyses, report both statistical significance and effect size, and consider power analysis during experimental design to ensure sufficient sample sizes for detecting biologically meaningful differences.

Comparative Analysis of INPPL1/SHIP-2 Antibodies for Various Research Applications