THTPA Human

What is THTPA and what is its primary function in human cells?

THTPA (Thiamine Triphosphatase) is a human protein involved in thiamine metabolism. The full protein sequence consists of 230 amino acids: "MAQGLIEVER KFLPGPGTEE RLQELGGTLE YRVTFRDTYY DTPELSLMQA DHWLRRREDS GWELKCPGAA GVLGPHTEYK ELTAEPTIVA QLCKVLRADG LGAGDVAAVL GPLGLQEVAS FVTKRSAWKL VLLGADEEEP QLRVDLDTAD FGYAVGEVEA LVHEEAEVPT ALEKIHRLSS MLGVPAQETA PAKLIVYLQR FRPQDYQRLL EVNSSRERPQ ETEDPDHCLG" . The protein functions as an enzyme that hydrolyzes thiamine triphosphate, playing a role in thiamine (vitamin B1) metabolism pathway regulation.

What methods are most effective for detecting THTPA expression in human samples?

Several complementary approaches offer robust detection of THTPA in human samples:

• Western Blotting: The rabbit polyclonal antibody ABIN528921 raised against full-length human THTPA protein offers high specificity for Western blotting applications . When optimizing this approach, researchers should use 12-15% gels appropriate for THTPA's size and include positive controls from tissues known to express the protein.

• Immunoprecipitation: The same rabbit polyclonal antibody (ABIN528921) can be used for immunoprecipitation experiments to isolate THTPA from complex samples .

• Immunohistochemistry: Mouse monoclonal (3F6) antibodies targeting AA 1-230 of THTPA are available for immunohistochemistry applications on paraffin-embedded sections .

• RNA Expression Analysis: Transcriptomic approaches can identify THTPA expression patterns across tissues and cell types, with methods like RT-PCR and RNA-Seq providing quantitative data on expression levels.

What is known about THTPA tissue distribution and expression patterns?

THTPA expression can be analyzed using the Human Protein Atlas resources, which provide comprehensive data on tissue and cellular distribution . The expression profile includes:

• Tissue-specific expression: The Human Protein Atlas offers RNA expression data across human tissues derived from HPA and GTEX datasets .

• Single-cell resolution: scRNA-seq data from normal tissues provides cell type-specific expression patterns, allowing categorization of THTPA as either cell type enriched, group enriched, cell type enhanced, or showing low cell type specificity .

• Expression clusters: THTPA has been grouped with genes showing similar expression patterns across tissues and cell types, with manual annotations describing common functional features and specificity .

What are key considerations when designing antibody-based experiments for THTPA?

When designing antibody-based experiments for THTPA research, consider these critical factors:

• Antibody selection: Multiple antibody options are available with different properties:

  • Rabbit polyclonal (ABIN528921): Targets full-length protein (AA 1-230), suitable for WB and IP

  • Mouse monoclonal (3F6): Targets AA 1-230, suitable for ELISA, WB, IHC

  • Various conjugated antibodies: HRP, biotin, and FITC-conjugated versions for specialized applications

• Epitope considerations: Different antibodies target distinct regions (full-length, N-terminal, specific amino acid ranges), affecting recognition of native versus denatured protein .

• Cross-reactivity: Some antibodies show reactivity with human only, while others recognize human, mouse, and rat THTPA, important for comparative studies .

• Controls: Include appropriate positive controls (recombinant THTPA) and negative controls (immunodepleted samples, isotype controls) to validate specificity.

• Application optimization: Each application (WB, IP, IHC, IF) requires specific optimization of antibody dilution, incubation conditions, and detection methods.

How should researchers approach THTPA functional studies?

Functional studies of THTPA require carefully designed experimental approaches:

• Enzymatic activity assays: Design assays that specifically measure thiamine triphosphate hydrolysis, distinguishing THTPA activity from other phosphatases.

• Genetic manipulation approaches:

  • Loss-of-function: CRISPR-Cas9 knockout or siRNA knockdown to study the effects of THTPA depletion

  • Gain-of-function: Overexpression of wild-type or mutant THTPA to assess functional consequences

• Interaction studies: Identify protein-protein interactions using techniques like co-immunoprecipitation with the available antibodies , proximity ligation assays, or yeast two-hybrid screening.

• Subcellular localization: Determine THTPA's localization within cells using fractionation methods or immunofluorescence with available antibodies.

• Metabolic impact assessment: Measure changes in thiamine metabolism when THTPA function is altered, potentially using mass spectrometry to quantify thiamine species.

What challenges exist in distinguishing THTPA activity from other phosphatases?

Distinguishing THTPA activity from other phosphatases presents several methodological challenges:

How can researchers optimize Western blotting protocols for THTPA detection?

Optimizing Western blotting for THTPA requires attention to several technical details:

• Sample preparation:

  • Lysis buffer selection: Use RIPA or NP-40 based buffers with protease inhibitors

  • Protein quantification: Load 20-50 μg total protein per lane

  • Denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer with DTT

• Gel electrophoresis and transfer:

  • Gel percentage: Use 12-15% polyacrylamide gels for optimal resolution

  • Transfer conditions: Semi-dry or wet transfer at 100V for 1 hour or 30V overnight

• Antibody selection and dilution:

  • Primary antibody: The rabbit polyclonal antibody ABIN528921 is validated for WB

  • Optimization: Perform antibody titration experiments to determine optimal dilution

  • Incubation: Overnight at 4°C in 5% BSA or milk in TBST

• Detection system:

  • HRP-conjugated secondary antibodies with ECL detection

  • Consider signal enhancement systems for low abundance detection

• Controls:

  • Positive control: Lysate from cells known to express THTPA

  • Loading control: β-actin or GAPDH for normalization

  • Molecular weight marker: To confirm the expected size (approximately 25 kDa)

What are the best approaches for THTPA immunoprecipitation experiments?

For successful immunoprecipitation of THTPA, researchers should follow these methodological guidelines:

• Antibody selection: The rabbit polyclonal antibody ABIN528921 is specifically validated for immunoprecipitation applications .

• Lysis conditions:

  • Buffer composition: Use mild lysis buffers (e.g., 1% NP-40, 150 mM NaCl, 50 mM Tris pH 7.4)

  • Protease inhibitors: Include complete protease inhibitor cocktail

  • Cell disruption: Gentle lysis to preserve native protein conformation

• Pre-clearing:

  • Incubate lysate with protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation before adding specific antibody

• Immunoprecipitation:

  • Antibody amount: Typically 2-5 μg per mg of total protein

  • Incubation time: Overnight at 4°C with gentle rotation

  • Bead type: Protein A beads for rabbit polyclonal antibodies

• Washing:

  • Buffer stringency: Balance between removing non-specific binding and maintaining specific interactions

  • Number of washes: Typically 3-5 washes with decreasing salt concentration

• Elution:

  • For Western blotting: Boiling in SDS sample buffer

  • For functional studies: Gentle elution with peptide competition or low pH

How should researchers analyze THTPA expression data from transcriptomic studies?

Analysis of THTPA expression data from transcriptomic studies requires rigorous statistical and bioinformatic approaches:

• Data normalization:

  • Between-sample normalization: TPM, FPKM, or appropriate count normalization methods

  • Batch effect correction: ComBat or similar algorithms if combining multiple datasets

• Statistical analysis:

  • Differential expression: t-tests, ANOVA, or non-parametric alternatives for comparing groups

  • Correlation analysis: Identify genes with similar expression patterns to THTPA

  • Multiple testing correction: Apply FDR or Bonferroni correction to control false positives

• Tissue/cell-type specificity analysis:

  • Calculate tissue specificity indices to quantify THTPA distribution

  • Apply enrichment metrics as used in the Human Protein Atlas

  • Compare against expression clusters of functionally related genes

• Visualization:

  • Heatmaps for cross-tissue or cross-condition comparison

  • Box plots or violin plots for expression level distribution

  • PCA or t-SNE plots for multivariate pattern visualization

• Biological interpretation:

  • Pathway analysis to contextualize THTPA expression changes

  • Co-expression network analysis to identify functional relationships

  • Integration with protein-level data when available

How can researchers address contradictory findings in THTPA research?

When encountering contradictory findings in THTPA research, apply these methodological strategies:

• Methodological comparison:

  • Carefully review experimental protocols, particularly antibody sources and specificities

  • Assess cell lines, culture conditions, and genetic backgrounds

  • Compare sample preparation methods that might affect THTPA detection

• Biological context evaluation:

  • Cell type differences: THTPA might function differently across cell types

  • Developmental stage: Consider temporal regulation of THTPA expression

  • Physiological state: Metabolic conditions might affect THTPA activity

• Systematic validation:

  • Replicate experiments using multiple complementary techniques

  • Use different antibodies targeting distinct epitopes of THTPA

  • Apply genetic approaches (knockout, knockdown) to confirm antibody specificity

• Computational reassessment:

  • Reanalyze raw data using standardized pipelines

  • Apply meta-analysis techniques to integrate multiple studies

  • Account for potential confounding variables

• Alternative hypotheses:

  • Consider post-translational modifications affecting detection or function

  • Evaluate potential isoforms or splice variants

  • Assess protein-protein interactions that might mask epitopes

What experimental approaches can address the relationship between THTPA and thiamine metabolism disorders?

Investigating THTPA's role in thiamine metabolism disorders requires multifaceted approaches:

• Cellular models:

  • THTPA knockout/knockdown in relevant cell types

  • Cell culture under thiamine-deficient conditions

  • Measurement of thiamine metabolites by HPLC or LC-MS/MS

• Clinical sample analysis:

  • THTPA expression in patient-derived samples

  • Genetic screening for THTPA variants in affected individuals

  • Correlation of THTPA levels with clinical parameters

• Enzymatic activity assessment:

  • Compare THTPA activity in normal versus pathological conditions

  • Evaluate the impact of disease-associated variants on enzymatic function

  • Measure substrate accumulation or product depletion

• Tissue-specific effects:

  • Analyze tissue distribution patterns from the Human Protein Atlas

  • Focus on high-expressing tissues for detailed functional studies

  • Investigate tissue-specific metabolic consequences of THTPA dysfunction

• Therapeutic modulation:

  • Test interventions that modify THTPA activity

  • Assess supplementation strategies that bypass metabolic blocks

  • Evaluate potential compensatory mechanisms

What new technologies are advancing THTPA research?

Emerging technologies offer new opportunities for THTPA research:

• CRISPR-Cas9 genome editing:

  • Precise knockout models in relevant cell lines

  • Introduction of specific mutations to study structure-function relationships

  • Base editing for specific amino acid substitutions without double-strand breaks

• Advanced structural biology:

  • Cryo-electron microscopy for high-resolution structural determination

  • AlphaFold or similar AI-based structure prediction tools

  • Computational modeling of substrate binding and catalysis

• Single-cell technologies:

  • Single-cell RNA-seq to identify cell-specific expression patterns

  • CyTOF or spectral flow cytometry for protein-level analysis

  • Spatial transcriptomics to map THTPA expression in tissue context

• Proteomics approaches:

  • Proximity labeling (BioID, APEX) to identify interaction partners

  • Global phosphoproteomics to assess THTPA regulation

  • Thermal proteome profiling to study ligand interactions

• Metabolomics integration:

  • Targeted metabolomics to measure thiamine species

  • Flux analysis using isotope-labeled precursors

  • Integration of metabolomic and transcriptomic data