lhpp Antibody

What is LHPP and why is it relevant to cancer research?

LHPP (Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase) is a 270 amino acid protein that functions as a homodimer and belongs to the HAD-like hydrolase superfamily. The protein plays a crucial role in regulating phosphate metabolism, which is essential for various cellular processes including energy production and signal transduction. LHPP has gained significant attention in cancer research due to its identified tumor suppressor role, particularly in hepatocellular carcinoma (HCC). Studies have shown that LHPP is downregulated in HCC tissues, and its higher expression correlates with better clinical outcomes for patients. The protein's ability to inhibit tumor cell growth and metastasis makes it an important target for cancer research and potential therapeutic development.

What types of LHPP antibodies are available for research applications?

LHPP antibodies are available in multiple formats to accommodate various experimental needs. Researchers can select from both monoclonal antibodies, such as the mouse monoclonal IgG2a kappa light chain antibody (B-2), and polyclonal antibodies, including rabbit polyclonal antibodies targeting specific regions like the C-terminus. These antibodies come in both non-conjugated forms and conjugated variants paired with detection molecules such as horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and various Alexa Fluor® conjugates. The availability of agarose-conjugated antibodies facilitates immunoprecipitation experiments. Different antibodies offer varying species reactivity profiles, with many recognizing LHPP across human, mouse, and rat samples, while some extend reactivity to additional species including cow, rabbit, guinea pig, horse, monkey, and pig.

How should researchers select the appropriate LHPP antibody for their specific experiment?

The selection of an appropriate LHPP antibody should be guided by several experimental considerations. First, determine your application requirements (western blotting, immunoprecipitation, immunofluorescence, or ELISA) as different antibodies demonstrate varying efficacy across these techniques. Consider the species of your experimental samples and ensure the antibody has validated reactivity for that species. For quantitative experiments, monoclonal antibodies like the LHPP Antibody (B-2) may provide more consistent results due to their recognition of a single epitope. When studying specific domains or investigating structure-function relationships, selecting an antibody targeting a specific region (such as C-terminal directed antibodies) can be advantageous. Finally, consider whether your experimental design requires a conjugated antibody (for direct detection) or an unconjugated primary antibody (for use with secondary detection systems). Always review validation data before purchase, especially regarding specificity testing in the cell lines or tissues most relevant to your research.

What are the optimal protocols for using LHPP antibodies in western blotting?

When using LHPP antibodies for western blotting, researchers should optimize several key parameters for successful detection. For sample preparation, cells should be lysed in RIPA buffer supplemented with protease inhibitors, with 20-50 μg of total protein loaded per lane. Given that LHPP is a 270 amino acid protein, a 10-12% SDS-PAGE gel provides optimal separation. After transfer to a PVDF or nitrocellulose membrane, blocking should be performed with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. For primary antibody incubation, dilute LHPP antibody according to manufacturer recommendations (typically 1:500-1:1000) in blocking buffer and incubate overnight at 4°C. After washing, apply an appropriate HRP-conjugated secondary antibody (unless using direct HRP-conjugated LHPP antibody) and develop using enhanced chemiluminescence. When investigating LHPP expression differences between normal and cancer cells, include positive controls such as normal liver cell line (LO2) which exhibits significantly higher LHPP expression compared to HCC cell lines like SMCC-7721, HepG2, Huh7, MHCC-97H, and LM3.

How can LHPP antibodies be effectively used in immunohistochemistry studies?

For effective immunohistochemistry using LHPP antibodies, tissue sections should be properly prepared through a series of methodical steps. Begin with 4 μm FFPE tissue sections mounted on glass slides. The deparaffinization process should use xylene followed by rehydration with graded alcohols. Block endogenous peroxidase activity by immersing slides in 3% H₂O₂ solution for 10 minutes. Antigen retrieval is critical and should be performed using 0.01 mol/L sodium citrate buffer (pH 6.0) with microwave heating for 10 minutes. After washing with PBS, block non-specific binding using 10% normal goat serum. Incubate sections with the primary LHPP antibody overnight at 4°C, using optimized dilutions (typically 1:100-1:200). Following PBS washes, apply appropriate secondary antibody for 30 minutes at room temperature and develop with diaminobenzidine (DAB) solution. Counter-stain with hematoxylin before dehydrating and mounting. Always include negative controls (primary antibody omitted) and positive controls (normal liver tissue) to ensure staining specificity. This protocol enables evaluation of LHPP expression patterns in tissue samples, which is particularly valuable for comparative studies between normal and tumor tissues.

What considerations are important when using LHPP antibodies for immunofluorescence?

When employing LHPP antibodies for immunofluorescence studies, several technical considerations can optimize experimental outcomes. For cell preparation, cultures should be grown on coverslips, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100. A critical blocking step using 3% BSA in PBS for 30 minutes helps minimize background signal. For primary antibody incubation, LHPP antibodies should be diluted according to manufacturer recommendations (typically 1:50-1:200) in blocking solution and applied for 1-2 hours at room temperature or overnight at 4°C. After washing, apply appropriate fluorophore-conjugated secondary antibodies (unless using direct fluorophore-conjugated LHPP antibodies) for 1 hour in the dark. Counterstain nuclei with DAPI and mount using anti-fade mounting medium. When examining subcellular localization, it's important to note that LHPP is predominantly expressed in liver and kidney with moderate expression in brain tissues, so appropriate controls should be selected. Consider co-staining with organelle markers to precisely determine subcellular localization. For quantitative analysis, standardize image acquisition parameters across all experimental conditions and analyze multiple fields per sample.

How do LHPP expression levels correlate with cancer progression and patient outcomes?

Research utilizing LHPP antibodies has revealed significant correlations between LHPP expression and cancer outcomes. Analysis of TCGA and Oncomine databases demonstrates that LHPP is consistently downregulated in hepatocellular carcinoma (HCC) tissues compared to adjacent normal tissues. More importantly, higher LHPP expression levels correlate positively with improved clinical outcomes in HCC patients. This inverse relationship between LHPP expression and cancer progression has been confirmed through multiple experimental approaches. Real-time PCR and western blot analyses using LHPP antibodies show significantly reduced LHPP expression across multiple HCC cell lines (SMCC-7721, HepG2, Huh7, MHCC-97H, and LM3) compared to normal liver cells (LO2). Gene set enrichment analysis (GSEA) of RNA-seq data from HCC patients reveals that low LHPP expression correlates with enrichment of cell cycle, mitotic spindle, and G2M checkpoint pathways, as well as metastasis-related gene signatures. These findings collectively establish LHPP as a promising diagnostic and prognostic biomarker for HCC, with potential applications in other cancer types.

What mechanisms underlie LHPP's tumor suppressor activity in HCC?

LHPP exerts its tumor suppressor effects through multiple molecular mechanisms that can be investigated using LHPP antibodies. Functional studies with lentivirus-mediated overexpression of LHPP in HCC cell lines (Huh7 and LM3) demonstrate that LHPP significantly inhibits cell viability, colony formation, migration, and invasiveness. At the molecular level, LHPP overexpression decreases the expression of several oncogenes including CCNB1 (involved in cell cycle regulation), PKM2 (a key glycolytic enzyme), and matrix metalloproteinases MMP7 and MMP9 (involved in invasion and metastasis). Real-time PCR analysis confirms negative correlations between LHPP and these oncogenes in human HCC tissues, with correlation coefficients of r = -0.34 (CCNB1), r = -0.37 (PKM2), r = -0.19 (MMP7), and r = -0.32 (MMP9). Gene set enrichment analysis (GSEA) of RNA-seq data from HCC patients further supports these findings, showing that low LHPP expression correlates with enrichment of cell cycle, mitotic spindle, and G2M checkpoint pathways. These combined mechanisms explain how LHPP constrains HCC cell growth and metastasis, establishing it as an important tumor suppressor in liver cancer.

How is LHPP regulated by m6A methylation and what are the implications for cancer metabolism?

Recent research has uncovered a critical regulatory mechanism involving m6A methylation of LHPP with significant implications for cancer metabolism. Studies in gastric cancer using RNA immunoprecipitation (RIP) with m6A-specific antibodies have revealed that LHPP mRNA undergoes m6A methylation, which affects its expression and function. This post-transcriptional modification appears to regulate LHPP's ability to alter cellular metabolism, particularly in cancer contexts. To investigate this mechanism, researchers established cell lines with LHPP overexpression or knockdown using lentiviral approaches in gastric cancer cells. Following stable transfection and puromycin selection (2 μg/ml), cells were subjected to detailed molecular analyses. Human phosphokinase array experiments comparing control and LHPP-overexpressing HGC-27 cells demonstrated that LHPP alters phosphorylation patterns of key metabolic regulators. The findings suggest that LHPP regulates cancer metabolism by influencing acetylation levels, creating a mechanistic link between m6A RNA methylation, LHPP expression, and metabolic reprogramming in cancer. This represents an emerging area where LHPP antibodies are essential tools for unraveling complex regulatory networks governing cancer metabolism.

What are common technical challenges when working with LHPP antibodies and how can they be addressed?

Researchers working with LHPP antibodies may encounter several technical challenges that require systematic troubleshooting. For western blotting applications, weak or absent signals may result from insufficient protein loading, antibody concentration issues, or ineffective antigen retrieval. Increasing protein load to 50-75 μg, optimizing antibody dilution (testing ranges from 1:200-1:1000), or employing enhanced antigen retrieval methods can resolve these issues. Background problems in immunohistochemistry or immunofluorescence can be addressed by extending blocking time (60-90 minutes), using alternative blocking agents (5% BSA, normal serum, or commercial blockers), and increasing wash steps. When comparing LHPP expression across different tissues or experimental conditions, inconsistent results may reflect biological variability or technical inconsistencies. Standardizing sample collection, processing times, and experimental conditions is essential. For antibody specificity concerns, validation experiments should include positive controls (normal liver tissue, which expresses high LHPP levels) and negative controls (antibody omission, pre-absorption with immunizing peptide). For co-immunoprecipitation experiments, optimize lysis conditions, antibody concentrations, and bead volumes to enhance protein capture while minimizing non-specific binding.

How should researchers design experiments to investigate the relationship between LHPP and oncogenes?

To effectively investigate LHPP's relationship with oncogenes, researchers should implement a comprehensive experimental design using LHPP antibodies. Begin with expression correlation studies using a panel of matched normal and cancer tissues or cell lines. Western blotting and qRT-PCR should quantify LHPP and target oncogene expression (focusing on CCNB1, PKM2, MMP7, and MMP9, which show established negative correlations with LHPP). Functional validation requires generating stable LHPP overexpression and knockdown cell models using lentiviral vectors, confirming manipulation efficiency with both western blot and qRT-PCR. These models should then be subjected to comprehensive phenotypic assays including proliferation assays (MTT/CCK-8), colony formation, migration (wound healing/transwell), and invasion assays. For mechanistic investigations, examine changes in oncogene expression following LHPP modulation using qRT-PCR and western blotting. Chromatin immunoprecipitation can determine if LHPP influences transcription factor binding to oncogene promoters. For in vivo validation, xenograft models with LHPP-modulated cells can assess tumor growth and metastasis, with immunohistochemistry using LHPP antibodies to confirm persistent expression changes throughout the experiment. This systematic approach allows robust characterization of LHPP's regulatory effects on oncogene networks.

What are the best practices for quantifying LHPP expression in clinical samples?

Accurate quantification of LHPP expression in clinical samples requires rigorous methodological approaches. For immunohistochemical analysis, tissue microarrays containing matched tumor and adjacent normal tissues provide efficient screening platforms. Staining should follow standardized protocols with consistent antibody dilutions, incubation times, and development conditions. Quantification should employ both intensity scoring (0-3+) and percentage of positive cells to calculate H-scores or Allred scores, with multiple independent pathologists performing blind assessments. For western blot quantification of LHPP in tissue lysates, equal protein loading (verified by housekeeping proteins like GAPDH or β-actin) is essential, with LHPP levels normalized to these controls. Densitometric analysis should be performed using software like ImageJ, with triplicate experiments for statistical validation. For mRNA analysis, careful RNA extraction followed by reverse transcription and qRT-PCR using validated primers provides transcript-level data, with normalization to multiple reference genes (e.g., GAPDH, ACTB, and HPRT1) improving reliability. Correlation analyses between LHPP expression and clinical parameters requires sufficient sample sizes with comprehensive clinical annotation, including tumor stage, grade, and patient outcome data. Statistical approaches should include appropriate tests (t-tests, ANOVA, correlation coefficients) with multiple comparison corrections and survival analyses (Kaplan-Meier, Cox regression).

How can LHPP antibodies be utilized in multi-parameter flow cytometry for cancer research?

Leveraging LHPP antibodies in multi-parameter flow cytometry opens new avenues for cancer research, particularly for analyzing heterogeneous cell populations and rare cancer stem cells. For optimal protocols, cells should be fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin or commercial permeabilization buffers to enable intracellular LHPP detection. LHPP antibodies conjugated to fluorophores with distinct emission spectra (PE, FITC, or Alexa Fluor® variants) allow simultaneous detection with other markers. For cancer stem cell identification, combine LHPP antibodies with established cancer stem cell markers (CD133, CD44, ALDH) and proliferation markers (Ki-67). When analyzing clinical samples, incorporate lineage-specific markers and viability dyes to exclude dead cells and non-relevant populations. Panel design must consider spectral overlap, requiring proper compensation controls. For quantitative analysis, include calibration beads to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF). This approach enables precise characterization of LHPP expression relative to cellular phenotype and function, potentially identifying subpopulations with differential LHPP expression that correlate with tumorigenic potential, treatment resistance, or metastatic capabilities.

What role does LHPP play in cancer metabolic reprogramming and how can this be investigated?

LHPP's emerging role in cancer metabolic reprogramming represents a frontier research area requiring sophisticated experimental approaches. Evidence suggests LHPP influences metabolic pathways critical for cancer cell adaptation and survival. To investigate this connection, researchers should establish stable cell lines with LHPP overexpression or knockdown in relevant cancer models, confirming modification with LHPP antibodies via western blotting. Metabolic profiling using techniques like Seahorse XF analysis can measure effects on glycolysis (extracellular acidification rate) and mitochondrial respiration (oxygen consumption rate). Liquid chromatography-mass spectrometry metabolomics should assess broad metabolite changes, particularly examining glycolytic intermediates, TCA cycle components, and amino acids. Given LHPP's negative correlation with PKM2 (pyruvate kinase M2, a key glycolytic enzyme) in HCC, enzyme activity assays for PKM2 and other metabolic enzymes should be performed, along with assessment of metabolic protein acetylation status which has been linked to LHPP function. Isotope tracing experiments using 13C-labeled glucose or glutamine can map carbon flux through metabolic pathways. For in vivo validation, PET imaging with 18F-FDG in LHPP-modulated xenograft models can assess glucose uptake differences. These comprehensive approaches can elucidate LHPP's role in rewiring cancer metabolism, potentially revealing novel therapeutic vulnerabilities.

How might LHPP interact with phosphohistidine signaling networks and what methodologies can explore this relationship?

The investigation of LHPP's interaction with phosphohistidine signaling networks represents a challenging frontier requiring specialized methodologies. As a phospholysine phosphohistidine inorganic pyrophosphate phosphatase, LHPP likely influences histidine phosphorylation, an understudied but important post-translational modification. Researchers should employ acid-labile phosphorylation-specific antibodies that can detect phosphohistidine residues, as traditional phosphoproteomic methods often miss these modifications due to their acid lability. Mass spectrometry protocols specifically adapted for histidine phosphorylation detection (using neutral or basic conditions) are essential. In vitro phosphatase assays using purified LHPP and synthetic phosphohistidine-containing peptides can determine direct enzymatic activity and substrate specificity. For cellular studies, generate LHPP knockout, knockdown, and overexpression models, then analyze global phosphohistidine levels using modified phosphoproteomic approaches. Proximity labeling methods like BioID or APEX2 fused to LHPP can identify proximal interacting proteins within signaling networks. For functional relevance, phosphomimetic and phospho-dead mutations of identified LHPP targets can assess biological consequences of phosphohistidine modification. Human phosphokinase arrays comparing control and LHPP-modulated cells can provide insights into broader signaling networks affected. These methodologies collectively enable exploration of LHPP's role in this emerging signaling paradigm with potential implications for cancer biology and therapeutic development.

Data Table: LHPP Expression in Cancer Cell Lines and Correlation with Oncogenes

*Expression determined by RT-PCR and Western Blot relative to normal liver cells