INPP5J Antibody

What is INPP5J and what are its primary functions in cellular processes?

INPP5J (also known as PIPP, PIB5PA, or INPP5) is an inositol polyphosphate-5-phosphatase that functions primarily in metabolic processes and signal transduction pathways. The human version of INPP5J has a canonical amino acid length of 1006 residues and a protein mass of 107.2 kilodaltons, with three identified isoforms. It is predominantly localized in the cytoplasm of cells . INPP5J catalyzes the dephosphorylation of various phosphoinositides, particularly PI(3,4,5)P3 and PI(4,5)P2, as well as inositol phosphates like Ins(1,3,4,5)P4 and Ins(1,4,5)P3 . These actions enable INPP5J to regulate various signaling cascades, particularly those downstream of growth factor receptors. Recent studies have implicated INPP5J in cancer biology, with evidence suggesting its depletion in MDA-MB-231 cells increases breast cancer cell transformation, pointing to potential tumor suppressor functions .

What types of INPP5J antibodies are available for research applications?

Multiple types of INPP5J antibodies are available, each optimized for specific experimental applications. Most commercially available options are polyclonal antibodies raised in rabbits, though other host species may be available . These antibodies target different epitopes of the INPP5J protein, including N-terminal regions, C-terminal regions, and internal amino acid sequences . The antibodies may be unconjugated or conjugated with various tags depending on the intended application. According to available data, most INPP5J antibodies demonstrate reactivity across multiple species including human, mouse, and rat samples, with some showing broader cross-reactivity to other mammalian species with varying degrees of homology (ranging from 79-100% predicted reactivity) . These antibodies have been validated for applications including Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunocytochemistry (ICC), and Enzyme-Linked Immunosorbent Assay (ELISA) .

How do I determine the optimal INPP5J antibody for my specific research question?

Selecting the optimal INPP5J antibody requires careful consideration of multiple experimental factors. First, determine your primary application (WB, IHC, IF, etc.) and ensure the antibody has been validated for that specific technique . Next, confirm species reactivity matches your experimental system, noting that sequence homology can vary significantly between species (e.g., mouse INPP5J shares approximately 79% homology with human INPP5J) . Consider epitope location when studying specific isoforms or domains—antibodies targeting N-terminal regions will detect different fragments than those targeting C-terminal regions . For interaction studies, choose antibodies that don't bind to domains involved in protein-protein interactions. Review validation data thoroughly, including positive and negative controls. For quantitative studies, select antibodies with demonstrated linear signal response. When investigating post-translational modifications, ensure your antibody doesn't have epitopes in regions that might be modified. Finally, consider conducting preliminary validation experiments with multiple antibodies to identify the most suitable option for your specific experimental conditions and cell/tissue types.

What are the optimal conditions for using INPP5J antibodies in Western blotting experiments?

For optimal Western blotting using INPP5J antibodies, prepare samples with phosphatase inhibitors to preserve the native phosphorylation state of INPP5J and related proteins. When resolving INPP5J, use 8-10% SDS-PAGE gels to effectively separate the 107.2 kDa protein . During transfer, employ lower current for longer duration (e.g., 30V overnight at 4°C) to ensure complete transfer of large proteins. For blocking, 5% non-fat dry milk in TBST is generally effective, though BSA may provide lower background for phospho-specific detection . The recommended antibody dilution ranges from 1:500 to 1:2000 for most INPP5J antibodies , but optimization may be necessary for your specific antibody and experimental conditions. Incubate primary antibody overnight at 4°C with gentle agitation to maximize specific binding while minimizing background. For detection, both chemiluminescence and fluorescence-based systems work well, though fluorescence may offer better quantification. Always include positive controls (e.g., cell lines known to express INPP5J) and negative controls (e.g., INPP5J-knockout samples if available). The expected molecular weight for detection is approximately 107 kDa, though variation may occur due to post-translational modifications or detection of specific isoforms .

How should INPP5J antibodies be used for effective immunohistochemistry and immunofluorescence studies?

For effective immunohistochemistry (IHC) and immunofluorescence (IF) using INPP5J antibodies, tissue fixation and antigen retrieval are critical first steps. For formalin-fixed paraffin-embedded (FFPE) tissues, citrate buffer (pH 6.0) heat-induced epitope retrieval typically yields optimal results. When using frozen sections, light fixation with 4% paraformaldehyde for 10-15 minutes is recommended to preserve epitope accessibility while maintaining tissue morphology . For blocking, use 5-10% normal serum from the same species as the secondary antibody to minimize non-specific binding. The recommended dilution for primary INPP5J antibodies ranges from 1:10 to 1:100 for IF/ICC and 1:50 to 1:100 for IHC , though optimization is necessary for specific antibodies. For IF applications, utilize confocal microscopy to detect the predominantly cytoplasmic localization of INPP5J . When performing co-localization studies, select secondary antibodies with minimal spectral overlap and include appropriate controls for autofluorescence and channel crosstalk. For IHC visualization, both DAB and AEC chromogens are compatible with INPP5J detection. Always include positive control tissues with known INPP5J expression and negative controls (either INPP5J-negative tissues or primary antibody omission) to validate staining specificity .

What considerations are important when using INPP5J antibodies for immunoprecipitation studies?

When conducting immunoprecipitation (IP) studies with INPP5J antibodies, several methodological considerations are essential for success. Begin by selecting antibodies specifically validated for IP applications, as not all INPP5J antibodies that work well in Western blotting or immunofluorescence will efficiently immunoprecipitate the native protein . Cell lysis conditions are critical—use mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions while effectively solubilizing membrane-associated INPP5J pools. Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) to maintain phosphorylation states of INPP5J and its interaction partners. Pre-clear lysates with Protein A/G beads to reduce non-specific binding. For antibody binding, overnight incubation at 4°C with gentle rotation typically yields optimal results. When probing for INPP5J interaction partners, consider crosslinking the antibody to beads to prevent heavy and light chain interference during subsequent Western blotting. For investigating phosphoinositide phosphatase activity in immunoprecipitates, include specialized buffers that maintain enzymatic function. Elution conditions should be carefully selected based on downstream applications—mild elution with peptide competition may preserve enzymatic activity for functional studies, while more stringent denaturing elution may be necessary for complete recovery. Always include isotype control antibodies as negative controls to identify non-specific precipitation.

How can I address non-specific binding when using INPP5J antibodies?

Non-specific binding is a common challenge when working with INPP5J antibodies that can compromise experimental interpretation. To address this issue, start with antibody selection—choose highly purified antibodies (affinity-purified options are preferable) that have been validated for your specific application. Optimize blocking conditions by testing different blocking agents (BSA, normal serum, commercial blockers) and concentrations (3-5% is typically effective). Adjust antibody dilutions—begin with manufacturer-recommended ranges (1:500-1:2000 for WB; 1:50-1:100 for IHC) and perform titration experiments to identify the optimal concentration that maximizes specific signal while minimizing background. For Western blotting applications, increase the stringency of wash steps by adding 0.1-0.3% Tween-20 to wash buffers and extending wash durations. When performing immunostaining, include a 0.3% Triton X-100 permeabilization step before antibody incubation to improve accessibility while reducing non-specific membrane binding. Pre-adsorption of the antibody with the immunizing peptide (if available) can serve as both a specificity control and potentially reduce non-specific binding. For tissues with high endogenous biotin or peroxidase activity, include specific blocking steps before antibody incubation. If cross-reactivity with other 5-phosphatases is suspected due to conserved domains, validate results with a second antibody targeting a different epitope or with genetic approaches (siRNA knockdown or CRISPR knockout controls).

What are the best practices for validating INPP5J antibody specificity in experimental systems?

Rigorous validation of INPP5J antibody specificity is essential for generating reliable research data. A comprehensive validation approach should include multiple complementary strategies. Begin with genetic controls—CRISPR/Cas9 knockout or siRNA knockdown of INPP5J followed by Western blotting to confirm disappearance or significant reduction of the detected band at the expected molecular weight (107.2 kDa) . Perform peptide competition assays using the specific immunogen peptide to block antibody binding, which should eliminate specific signals while leaving non-specific signals intact . When possible, employ orthogonal detection methods by comparing results using multiple antibodies targeting different epitopes of INPP5J. For immunostaining applications, co-localization studies with GFP-tagged INPP5J can confirm antibody specificity. Phosphatase assays can provide functional validation—immunoprecipitated INPP5J should demonstrate activity against its known substrates, particularly PI(3,4,5)P3 and PI(4,5)P2 . Cross-reactivity assessment is important—test the antibody against recombinant proteins of closely related 5-phosphatases to ensure specificity. Technique-specific validation is also essential—an antibody that works well for Western blotting may not be suitable for immunoprecipitation or immunohistochemistry. For quantitative applications, establish a standard curve using recombinant INPP5J to confirm linear signal response. Finally, compare your findings with published literature to ensure consistency with established INPP5J expression patterns and molecular weight .

How can I optimize detection of low-abundance INPP5J in various tissue samples?

Detecting low-abundance INPP5J in tissue samples requires specialized approaches to enhance sensitivity while maintaining specificity. Begin by optimizing sample preparation—for protein extraction, use buffers containing 1% NP-40 or Triton X-100 with protease and phosphatase inhibitors to maximize INPP5J recovery while preserving its native state. Consider protein concentration methods such as TCA precipitation or immunoprecipitation to enrich for INPP5J before analysis. For Western blotting, employ high-sensitivity detection systems—fluorescent secondary antibodies often provide 2-3 fold greater sensitivity than traditional chemiluminescence. Signal amplification strategies can significantly enhance detection limits—tyramide signal amplification (TSA) can increase sensitivity up to 100-fold for immunohistochemistry and immunofluorescence applications . When using immunohistochemistry, optimize antigen retrieval methods—test both heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0) and enzymatic retrieval to determine which best exposes INPP5J epitopes in your specific tissue. For immunofluorescence, confocal microscopy with increased photomultiplier gain settings and longer exposure times can detect weaker signals, while specialized techniques like Airyscan or stimulated emission depletion (STED) microscopy offer even greater sensitivity. Consider using amplification-based molecular techniques like RNAscope to detect INPP5J mRNA as a complementary approach to protein detection. Always include positive control samples with known INPP5J expression to validate your detection protocol and establish the detection threshold for your specific experimental system .

How can INPP5J antibodies be utilized to study phosphoinositide signaling pathways in cancer models?

INPP5J antibodies provide powerful tools for investigating phosphoinositide signaling disruptions in cancer. For studying INPP5J's tumor suppressor functions, use validated antibodies in immunohistochemistry to evaluate expression patterns across tumor progression stages and correlate with clinical outcomes . Combine this with phospho-specific antibodies against AKT and other PI3K pathway components to assess the impact of INPP5J expression on downstream signaling. Multi-color immunofluorescence with confocal microscopy can reveal spatial relationships between INPP5J and its substrates, particularly PI(3,4,5)P3, using specific lipid-binding domain probes. For mechanistic studies, immunoprecipitation with INPP5J antibodies followed by mass spectrometry can identify novel interaction partners in cancer cells versus normal cells. Phosphatase activity assays using immunoprecipitated INPP5J from various cancer models can determine if catalytic activity is altered in malignancy. To investigate INPP5J regulation, combine chromatin immunoprecipitation (ChIP) of transcription factors with INPP5J expression analysis to elucidate transcriptional control mechanisms. Live-cell imaging using INPP5J antibodies conjugated to cell-permeable peptides can monitor dynamic changes in INPP5J localization during cancer cell migration and invasion. Importantly, use INPP5J antibodies in combination with genetic approaches (CRISPR/Cas9 knockout or overexpression) to validate phenotypic effects observed in cancer models, particularly regarding the observation that INPP5J depletion increases breast cancer cell transformation in MDA-MB-231 cells .

What methodologies can be employed to study INPP5J phosphatase activity using specific antibodies?

Studying INPP5J phosphatase activity requires specialized approaches combining antibody-based techniques with enzymatic assays. Begin with immunoprecipitation using validated INPP5J antibodies to isolate the enzyme from cell or tissue lysates . For the immunoprecipitation, use non-ionic detergent buffers (0.5-1% NP-40) that preserve enzymatic activity. After isolation, assess phosphatase activity by incubating the immunoprecipitates with radiolabeled substrates (particularly 32P-labeled PI(3,4,5)P3 or PI(4,5)P2) and measure released phosphate by thin-layer chromatography or liquid scintillation counting . Alternatively, employ fluorescent phosphoinositide analogs with FRET-based detection systems for real-time activity monitoring. For higher throughput analysis, malachite green assays can quantify released phosphate from non-radioactive substrates. To assess INPP5J activity in intact cells, combine antibody-based detection of INPP5J with live-cell imaging of fluorescent PI(3,4,5)P3 biosensors (PH domains fused to fluorescent proteins). Structure-function studies can be performed by comparing wild-type INPP5J activity with mutant forms using site-directed mutagenesis of catalytic residues. For studying regulation of INPP5J activity, analyze phosphorylation status using phospho-specific antibodies against known regulatory sites, followed by correlation with enzymatic activity measurements. Always include appropriate controls: heat-inactivated samples, catalytically inactive INPP5J mutants, and competitive inhibitors of inositol phosphatases like general phosphatase inhibitors (orthovanadate) or specific inhibitors where available.

How can INPP5J antibodies be utilized to investigate protein-protein interactions in signal transduction networks?

INPP5J antibodies provide versatile tools for exploring protein-protein interactions within signaling networks. Co-immunoprecipitation (co-IP) using INPP5J antibodies is the foundation for identifying interaction partners—use mild lysis conditions (0.5-1% NP-40 or Triton X-100) to preserve protein complexes and include phosphatase inhibitors to maintain phosphorylation-dependent interactions . Following immunoprecipitation, mass spectrometry analysis can identify novel binding partners. Proximity ligation assay (PLA) offers a powerful approach for visualizing INPP5J interactions in situ—combine INPP5J antibodies with antibodies against suspected interaction partners to generate fluorescent signals only when proteins are within 40nm of each other. For investigating dynamic interactions, use FRET-based approaches with labeled antibody fragments. Bimolecular fluorescence complementation (BiFC) can validate direct interactions by fusing potential partners with complementary fluorescent protein fragments. To map interaction domains, perform pull-down assays using antibodies against different INPP5J domains or truncation mutants. For high-throughput screening, antibody arrays containing INPP5J antibodies can capture interaction partners from complex lysates. Blue-native PAGE combined with Western blotting using INPP5J antibodies can preserve and detect native protein complexes. When investigating interactions with membrane components or lipid rafts, combine detergent-resistant membrane fractionation with INPP5J immunoblotting. For temporal dynamics, synchronize cells at different cell cycle phases or stimulate with growth factors for various durations before performing co-IP with INPP5J antibodies. Always validate interactions using reciprocal co-IP and controls for non-specific binding, such as isotype control antibodies and competitive peptide blocking .

How do expression patterns of INPP5J compare across different tissue types and disease states?

INPP5J expression demonstrates significant tissue-specific and disease-state variation that requires careful analysis. In normal tissues, INPP5J is predominantly expressed in the cytoplasm of cells across various tissue types . Based on available research findings, the expression patterns show differences across organ systems that correlate with tissue-specific phosphoinositide signaling requirements. In pathological conditions, particularly in cancer, INPP5J expression patterns appear to undergo significant alterations. Research indicates that INPP5J may function as a tumor suppressor in certain contexts, as depletion of INPP5J in MDA-MB-231 breast cancer cells leads to increased cellular transformation . This suggests that decreased INPP5J expression may contribute to cancer progression in some tumor types. When analyzing INPP5J expression data, researchers should consider several factors: first, ensure antibody specificity through appropriate controls; second, distinguish between changes in protein levels versus altered subcellular localization; third, correlate expression with relevant clinical parameters when studying disease tissues; fourth, consider potential post-translational modifications that might affect antibody recognition; and fifth, compare findings across multiple detection methods (immunohistochemistry, Western blotting, qPCR) to validate observations. For quantitative expression analysis, digital pathology approaches with standardized scoring systems can provide more objective assessments of INPP5J expression patterns across different tissue types and disease states.

What are the key considerations when interpreting contradictory results between different INPP5J antibodies?

When confronted with contradictory results between different INPP5J antibodies, a systematic analytical approach is essential. Begin by examining epitope differences—antibodies targeting distinct regions of INPP5J (N-terminal, C-terminal, or internal domains) may detect different isoforms or post-translationally modified variants . Review validation data for each antibody, including Western blot images demonstrating specificity and the presence of single versus multiple bands. Assess cross-reactivity profiles—some antibodies may detect related phosphatases due to conserved domains within the 5-phosphatase family. Consider the technological context—an antibody optimized for denatured proteins (Western blotting) may perform poorly with native proteins (immunoprecipitation) and vice versa. Evaluate species cross-reactivity—sequence differences between human, mouse, and rat INPP5J may affect antibody performance despite predicted reactivity . Investigate sample preparation variables—fixation methods, antigen retrieval techniques, and buffer compositions can significantly impact epitope accessibility. For resolution, perform side-by-side comparisons under identical conditions using positive controls (overexpression systems) and negative controls (INPP5J-knockout samples). Employ orthogonal methods like mass spectrometry or RNA analysis to validate protein identification. When publishing results with INPP5J antibodies, transparently report all antibody details (catalog number, lot, dilution, incubation conditions) and validation methods to enable proper interpretation and reproducibility. If contradictions persist, consider that both results may be correct but reflect different aspects of INPP5J biology, such as context-dependent expression of isoforms or post-translational modifications.

How can researchers effectively compare phosphatase activity data across different experimental systems using INPP5J antibodies?

Comparing INPP5J phosphatase activity across experimental systems requires careful methodological standardization and data normalization. First, establish consistent immunoprecipitation conditions using the same INPP5J antibody across all systems to ensure comparable enzyme isolation . Standardize substrate preparation—use defined concentrations of the same substrate (preferably purified PI(3,4,5)P3 or PI(4,5)P2) with consistent specific activity for radioactive assays . Implement internal controls by including a standard sample of recombinant INPP5J with known activity in each experimental batch to account for day-to-day variations. For normalization, quantify the amount of immunoprecipitated INPP5J in each sample by Western blotting and express activity per unit of enzyme rather than per total protein. Consider reaction kinetics—perform time-course experiments to ensure measurements are taken within the linear range of the reaction. Account for system-specific factors that might influence INPP5J activity, such as endogenous inhibitors or activators that co-immunoprecipitate with INPP5J. For cross-laboratory comparisons, develop standard units of INPP5J activity based on reference standards. When comparing activity across different cell types or tissues, complement biochemical assays with in situ approaches like the use of fluorescent phosphoinositide biosensors to measure INPP5J activity in intact cells. For data interpretation, contextualize activity measurements with expression levels and localization data to distinguish between changes in specific activity versus alterations in enzyme abundance. Finally, consider developing mathematical models that integrate multiple parameters (substrate availability, enzyme concentration, reaction conditions) to facilitate meaningful comparisons across diverse experimental systems.