THTPA Antibody

What is the molecular identity of THTPA and why is it significant for antibody development?

Thiamine triphosphatase (THTPA) is a 25.566 kDa enzyme belonging to the ThTPase protein family that catalyzes the hydrolysis of thiamine triphosphate to thiamine diphosphate and inorganic phosphate. The protein is encoded by the THTPA gene (gene ID: 79178) located on human chromosome 14 and demonstrates considerable conservation across species . The significance of THTPA in neural metabolism and thiamine homeostasis has prompted the development of targeted antibodies for studying its expression, localization, and functional role in various tissues. The protein's calculated molecular weight is 26 kDa, though it often appears between 26-35 kDa on Western blots due to post-translational modifications . The development of specific antibodies against THTPA has enabled researchers to investigate thiamine metabolism in both normal physiological conditions and disease states, particularly in neurological and metabolic disorders where thiamine pathways may be disrupted.

How do polyclonal and monoclonal THTPA antibodies differ in research applications?

Polyclonal THTPA antibodies, such as the rabbit-derived 15486-1-AP and HPA028876, recognize multiple epitopes on the THTPA protein, offering advantages in signal amplification and detection sensitivity across various applications . These antibodies typically provide robust signals in Western blot, immunohistochemistry, and immunofluorescence applications due to their ability to bind multiple epitopes simultaneously. Conversely, monoclonal antibodies like the mouse OTI13E3 clone target a single epitope with high specificity, making them particularly valuable for distinguishing between closely related proteins or when absolute specificity is required . The selection between these antibody types should be guided by experimental requirements:

The choice between polyclonal and monoclonal antibodies should be determined by whether the experimental design prioritizes sensitivity (polyclonal) or specificity (monoclonal), particularly in complex tissue samples where cross-reactivity might be problematic .

What validation strategies are essential before employing a new THTPA antibody in a research project?

Comprehensive validation of THTPA antibodies is critical for ensuring experimental reliability. A methodical validation approach should include:

• Western blot analysis: Verify the antibody detects a band of appropriate molecular weight (26-35 kDa for THTPA) in relevant cell lysates. The 15486-1-AP antibody has been validated in multiple cell lines including K-562, A431, HeLa, and PC-3 cells, as well as in tissue extracts from human brain, mouse testis, and mouse uterus .

• Positive and negative controls: Include lysates from cells known to express THTPA (positive control) and those with THTPA knockdown or from tissues not expressing the protein (negative control).

• Cross-species reactivity assessment: If working with non-human models, verify antibody reactivity as demonstrated with antibodies like 15486-1-AP which shows reactivity with human, mouse, and rat samples .

• Comparison across applications: Confirm antibody performance in multiple detection methods. For example, the 15486-1-AP antibody has been validated for Western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence applications .

• Immunogen sequence analysis: Review the immunogen used to generate the antibody for potential cross-reactivity with related proteins. The Sigma HPA028876 antibody, for instance, was developed using a specific immunogen sequence (GAAGVLGPHTEYKELTAEPTIVAQLCKVLRADGLGAGDVAAVLGPLGLQEVASFVTKRSAWKLVLLGADEEEPQLRVDLDTADFGYAVGEVEALVHEEAEVPTALEKIHRLSSMLGVP) .

Only after comprehensive validation should researchers proceed with experimental applications, as this minimizes the risk of artifactual results that could arise from non-specific binding or cross-reactivity issues.

What are the optimized protocols for successful Western blot detection of THTPA?

Successful Western blot detection of THTPA requires careful optimization of multiple parameters. Based on the manufacturer recommendations and validation data, the following protocol is recommended:

• Sample preparation: Extract proteins from cells or tissues using standard lysis buffers containing protease inhibitors. THTPA has been successfully detected in K-562, A431, HeLa, and PC-3 cells, as well as in tissue extracts from human brain, mouse testis, and mouse uterus .

• Protein loading and separation: Load 5-20 μg of total protein per lane on 10-12% SDS-PAGE gels, as THTPA has a molecular weight of approximately 26 kDa but may appear between 26-35 kDa due to post-translational modifications .

• Transfer conditions: Use PVDF membrane and standard wet transfer protocols (100V for 60-90 minutes).

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute THTPA antibodies according to manufacturer recommendations:

  • For polyclonal antibody 15486-1-AP: Use 1:500-1:1000 dilution

  • For monoclonal antibody OTI13E3 (M03875): Use 1:2000 dilution

  • For Sigma HPA028876: Use 0.04-0.4 μg/mL

• Detection: Apply appropriate secondary antibodies and visualize using chemiluminescence or infrared detection systems.

For troubleshooting purposes, it's important to note that THTPA may undergo post-translational modifications resulting in multiple bands or shifts in apparent molecular weight from the calculated 26 kDa to the observed 26-35 kDa range .

How should THTPA antibodies be optimized for immunohistochemical applications?

Optimizing THTPA antibodies for immunohistochemistry requires careful attention to fixation, antigen retrieval, and antibody dilution:

• Tissue fixation and processing: Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used, with fixation times of 24-48 hours recommended for optimal antigen preservation.

• Sectioning: Prepare 4-6 μm thick sections for optimal antibody penetration and signal detection.

• Antigen retrieval: This is critical for THTPA detection, with specific recommendations:

  • For antibody 15486-1-AP: Use TE buffer at pH 9.0 as primary method; alternatively, citrate buffer pH 6.0 can be used

  • For other antibodies, heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

• Blocking: Apply 3-5% normal serum (matching the species of the secondary antibody) to reduce non-specific binding.

• Primary antibody dilution:

  • For 15486-1-AP: Use 1:20-1:200 dilution

  • For Abbexa THTPA antibody: Use 1:20-1:200 dilution

  • For Sigma HPA028876: Use 1:200-1:500 dilution

• Incubation conditions: Optimal results are typically achieved with overnight incubation at 4°C, though 1-2 hours at room temperature may be sufficient for some antibodies.

• Detection system: HRP-conjugated secondary antibodies with DAB substrate are commonly used, with hematoxylin counterstaining to visualize cellular morphology.

Positive control tissues should include human stomach cancer tissue, which has been validated for THTPA antibody 15486-1-AP . The antibody titers should be optimized for each tissue type and fixation method, as antibody performance can vary significantly between tissue sources.

What strategies effectively minimize background and non-specific binding in immunofluorescence with THTPA antibodies?

Minimizing background and non-specific binding is particularly important for immunofluorescence applications with THTPA antibodies. The following strategies are recommended based on validated protocols:

• Cell fixation optimization: For cultured cells like HepG2 (validated for 15486-1-AP antibody), use 4% paraformaldehyde for 15-20 minutes at room temperature, followed by permeabilization with 0.1-0.3% Triton X-100 for 5-10 minutes .

• Blocking optimization:

  • Extend blocking time to 1-2 hours with 5-10% normal serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 to blocking buffer to improve antibody penetration

  • Include 1-2% BSA to reduce non-specific protein interactions

• Antibody dilution optimization:

  • For 15486-1-AP: Use 1:10-1:100 dilution range, with titration recommended for each cell type

  • For Abbexa THTPA antibody: Use 1:20-1:200 dilution

• Primary antibody incubation: Extending incubation time to overnight at 4°C can improve specific signal while reducing background.

• Wash optimization: Perform extensive washing (5-6 times, 5 minutes each) with PBS containing 0.05-0.1% Tween-20 after both primary and secondary antibody incubations.

• Secondary antibody considerations: Use highly cross-adsorbed secondary antibodies to minimize cross-species reactivity, and include 1% BSA in secondary antibody dilution buffer.

• Autofluorescence reduction: When working with tissues or certain cell types, pretreat with 0.1% Sudan Black B in 70% ethanol for 20 minutes to reduce autofluorescence.

• Negative controls: Always include negative controls (primary antibody omission, isotype controls, or pre-immune serum) to assess background levels and non-specific binding.

These optimization strategies should be systematically tested and modified according to the specific cell type or tissue being studied, as THTPA detection sensitivity can vary considerably between different biological samples.

Why might multiple bands or unexpectedly shifted bands appear in Western blots when using THTPA antibodies?

The appearance of multiple bands or band shifts when using THTPA antibodies can be attributed to several biological and technical factors:

• Post-translational modifications: THTPA has a calculated molecular weight of 26 kDa but is frequently observed between 26-35 kDa on Western blots due to various post-translational modifications . These modifications may include:

  • Phosphorylation: THTPA contains several potential phosphorylation sites that can affect protein mobility

  • Glycosylation: Limited N-glycosylation sites may contribute to higher molecular weight bands

  • Ubiquitination: Potential ubiquitination may result in ladder-like patterns of higher molecular weight bands

• Splice variants: Alternative splicing of the THTPA gene may produce protein isoforms of different molecular weights, resulting in multiple specific bands.

• Proteolytic degradation: Incomplete protease inhibition during sample preparation can result in THTPA degradation products, typically appearing as lower molecular weight bands.

• Cross-reactivity: Polyclonal antibodies, in particular, may recognize epitopes present in related proteins, especially other phosphatases with structural similarity to THTPA.

• Sample preparation variables: Reducing conditions, heating time, and buffer composition can all affect protein migration patterns.

To distinguish between these possibilities, researchers should:

• Compare results across multiple THTPA antibodies (e.g., both 15486-1-AP and OTI13E3)

• Perform peptide competition assays to confirm specificity

• Analyze lysates from THTPA-knockout or knockdown cells as negative controls

• Use phosphatase or glycosidase treatments to determine if post-translational modifications are responsible for band shifts

The observation that the 15486-1-AP antibody consistently detects bands in the 26-35 kDa range across multiple validated cell lines (K-562, A431, HeLa, PC-3) and tissues (human brain, mouse testis, mouse uterus) suggests that this pattern reflects authentic THTPA detection rather than non-specific binding .

How can contradictory results between immunohistochemistry and Western blot be reconciled when studying THTPA expression?

Reconciling contradictory results between immunohistochemistry (IHC) and Western blot (WB) when studying THTPA requires systematic analysis of several potential factors:

• Epitope accessibility differences:

  • In WB, proteins are denatured, exposing all epitopes

  • In IHC, protein folding and interactions may mask certain epitopes

  • Solution: Try multiple antibodies targeting different THTPA epitopes, such as comparing results between 15486-1-AP and OTI13E3

• Fixation-induced epitope alterations:

  • Formalin fixation can create protein cross-links that alter epitope structure

  • Solution: Optimize antigen retrieval methods, comparing both TE buffer pH 9.0 and citrate buffer pH 6.0 as recommended for 15486-1-AP

• Threshold detection differences:

  • WB may detect low levels of expression not visible in IHC

  • Solution: Use more sensitive detection systems for IHC, such as tyramide signal amplification

• Sample preparation variables:

  • Cell lysis methods for WB may extract proteins differently than those preserved in tissue sections

  • Solution: Prepare samples for both methods from the same tissue source simultaneously

• Antibody concentration optimization:

  • Different optimal dilutions are needed for each method:

    • For 15486-1-AP: WB (1:500-1:1000) vs. IHC (1:20-1:200)

    • For Abbexa antibody: WB (1:500-1:2000) vs. IHC (1:20-1:200)

    • For Sigma HPA028876: WB (0.04-0.4 μg/mL) vs. IHC (1:200-1:500)

• Validation approach:

  • Perform cell type-specific knockdown of THTPA and evaluate both methods

  • Use complementary techniques like in situ hybridization to detect THTPA mRNA

  • Compare results across multiple antibodies and detection systems

When faced with contradictory results, researchers should systematically document the precise experimental conditions for both methods, including antibody lot numbers, dilutions, incubation times, and detection systems. This information is crucial for troubleshooting and determining whether the discrepancies reflect technical limitations or genuine biological complexity in THTPA expression and localization.

What considerations are critical when using THTPA antibodies across different species models?

Using THTPA antibodies across different species models requires careful consideration of several factors to ensure reliable and interpretable results:

• Sequence homology analysis:

  • Human THTPA shares approximately 85% amino acid sequence identity with mouse and rat orthologs

  • The 15486-1-AP and Abbexa antibodies have been validated for reactivity with human, mouse, and rat samples

  • The OTI13E3 monoclonal antibody is specifically validated only for human samples

  • The Sigma HPA028876 antibody is indicated for human reactivity only

• Epitope conservation verification:

  • Analyze the immunogen sequence used to generate the antibody against the target species sequence

  • For example, the Sigma HPA028876 antibody immunogen sequence should be aligned with potential target species sequences to predict cross-reactivity

• Application-specific validation:

  • Even antibodies labeled for cross-species reactivity may perform differently across applications

  • The 15486-1-AP antibody has been specifically validated for detecting THTPA in:

    • Human samples: K-562, A431, HeLa, PC-3 cells, human brain tissue, human stomach cancer tissue

    • Mouse samples: mouse testis tissue, mouse uterus tissue

• Dilution optimization by species:

  • Optimal antibody dilutions often differ between species due to variations in epitope accessibility and binding affinity

  • Start with the recommended dilution range and perform careful titration for each new species

• Controls for cross-species applications:

  • Include species-specific positive controls (tissues known to express THTPA)

  • When possible, include knockout/knockdown controls in the specific species being studied

  • Consider peptide competition assays using species-specific recombinant THTPA

• Detection system considerations:

  • Secondary antibodies must be specifically validated against the primary antibody host species (rabbit for 15486-1-AP, HPA028876, and Abbexa antibody; mouse for OTI13E3)

  • When working with tissues that have high endogenous biotin or peroxidase activity, special blocking steps may be required

When planning cross-species studies, researchers should conduct preliminary validation experiments to confirm antibody performance in each species of interest, rather than assuming cross-reactivity based on manufacturer claims alone.

How have THTPA antibodies contributed to understanding thiamine metabolism in neurological disorders?

THTPA antibodies have made significant contributions to understanding altered thiamine metabolism in neurological disorders through several important research applications:

• Expression pattern analysis in brain regions:

  • THTPA antibodies have enabled the mapping of thiamine triphosphatase expression across different brain regions, revealing differential expression patterns that may correlate with vulnerability to thiamine deficiency disorders

  • The 15486-1-AP antibody has been validated for human brain tissue analysis by Western blot, allowing quantitative comparison of THTPA expression across brain regions and in disease states

• Cellular and subcellular localization studies:

  • Immunofluorescence using THTPA antibodies has revealed the subcellular distribution of this enzyme in neurons and glial cells

  • This has enhanced understanding of compartmentalized thiamine metabolism within different cell types in the nervous system

• Neurodegenerative disease investigations:

  • THTPA antibody-based studies have examined alterations in thiamine metabolism in conditions including:

    • Alzheimer's disease: where thiamine metabolism disturbances may contribute to neurodegeneration

    • Wernicke-Korsakoff syndrome: a thiamine deficiency disorder with characteristic neurological symptoms

    • Parkinson's disease: where metabolic dysfunction may include thiamine pathway abnormalities

• Developmental neurobiology applications:

  • Immunohistochemical analysis of THTPA expression during brain development has provided insights into the role of thiamine metabolism in neuronal maturation and circuit formation

• Response to metabolic stress:

  • Studies using THTPA antibodies have examined how neurons regulate thiamine triphosphate levels under conditions of oxidative stress, hypoxia, and glucose deprivation

  • These investigations have revealed potential adaptive mechanisms involving THTPA regulation

The specificity of 15486-1-AP for detecting THTPA in human brain tissue by Western blot and IHC has been particularly valuable in these studies . Additionally, the ability to detect THTPA in mouse brain tissues with the same antibody has facilitated translational research between mouse models and human neurological conditions. Future directions include further characterization of THTPA's role in neurodegenerative disease progression and the potential for targeting thiamine metabolism pathways therapeutically.

What techniques combine THTPA antibodies with other molecular tools to study enzyme functionality?

Researchers have developed sophisticated approaches that combine THTPA antibodies with other molecular tools to comprehensively study enzyme functionality:

These integrated approaches leverage the specificity of well-validated THTPA antibodies while overcoming the limitations of any single technique. The combined methods provide comprehensive insights into THTPA function in various cellular contexts and disease states.

How can researchers distinguish between specific and non-specific signals when studying low-abundance THTPA expression?

Distinguishing between specific and non-specific signals is particularly challenging when studying low-abundance proteins like THTPA. Researchers can implement several rigorous strategies to ensure reliable detection:

• Multiple antibody validation approach:

  • Compare results from different antibodies targeting distinct THTPA epitopes:

    • Polyclonal antibody 15486-1-AP (rabbit)

    • Monoclonal antibody OTI13E3 (mouse)

    • Abbexa polyclonal antibody (rabbit)

    • Sigma HPA028876 (rabbit)

  • Convergent results from antibodies with different properties provide stronger evidence for specific detection

• Genetic knockdown/knockout controls:

  • Generate THTPA knockdown/knockout cells using siRNA, shRNA, or CRISPR-Cas9

  • Compare antibody signals between wild-type and knockdown/knockout samples

  • True specific signals should be significantly reduced or eliminated in knockdown/knockout samples

• Peptide competition assays:

  • Pre-incubate the THTPA antibody with excess purified THTPA protein or immunogenic peptide

  • Apply the pre-absorbed antibody to samples in parallel with the standard antibody

  • Specific signals should be substantially reduced in the pre-absorbed condition

• Signal amplification with validation:

  • For low-abundance detection, employ signal amplification methods like:

    • Tyramide signal amplification for immunohistochemistry/immunofluorescence

    • Enhanced chemiluminescence for Western blot

  • Always run appropriate controls to distinguish amplified specific signals from amplified background

• Quantitative analysis parameters:

  • Establish clear signal-to-background ratio thresholds for positive detection

  • Use digital image analysis to quantify signals objectively

  • Apply statistical methods to determine if signals significantly exceed background levels

• Complementary technique verification:

  • Confirm protein expression using non-antibody-based methods:

    • mRNA detection via RT-qPCR or in situ hybridization

    • Mass spectrometry-based proteomics

    • Functional assays measuring thiamine triphosphatase activity

• Optimized detection protocols:

  • For Western blot: Load higher protein amounts (20-50 μg), use high-sensitivity substrates, and optimize exposure times

  • For immunohistochemistry/immunofluorescence: Use higher antibody concentrations within the recommended ranges (e.g., 1:20-1:50 for 15486-1-AP) , extend incubation times, and optimize antigen retrieval

By implementing these complementary approaches, researchers can substantially increase confidence in the specificity of THTPA detection, even in samples with low expression levels or in challenging experimental conditions.

What emerging research directions are THTPA antibodies enabling in metabolic and neurological disease studies?

THTPA antibodies are instrumental in advancing several emerging research directions at the intersection of thiamine metabolism, neurological function, and metabolic disease:

• Neurodegenerative disease mechanisms: Well-validated THTPA antibodies like 15486-1-AP are enabling detailed investigations into the role of disturbed thiamine metabolism in Alzheimer's disease, Parkinson's disease, and other neurodegenerative conditions . These studies are revealing how alterations in THTPA expression and activity may contribute to neuronal vulnerability and disease progression.

• Metabolic stress responses: Research using THTPA antibodies is uncovering how cells modulate thiamine triphosphate levels during various metabolic stresses, including hypoxia, oxidative stress, and nutrient deprivation. These studies suggest THTPA may function as a metabolic sensor and regulator in stress adaptation pathways.

• Cancer metabolism: The validation of THTPA antibodies in cancer cell lines (K-562, A431, HeLa, PC-3) and cancer tissues (human stomach cancer) is facilitating investigations into the role of thiamine metabolism in cancer progression and therapy resistance . These studies may identify novel metabolic vulnerabilities that could be therapeutically targeted.

• Thiamine-responsive disorders: THTPA antibody-based research is enhancing understanding of rare genetic disorders characterized by abnormal responses to thiamine supplementation, potentially leading to improved diagnostic approaches and treatment strategies.

• Aging and metabolic decline: Studies examining age-related changes in THTPA expression and activity across tissues are providing insights into how thiamine metabolism may contribute to metabolic decline during aging.

• Drug development and screening: THTPA antibodies are enabling high-throughput screening approaches to identify compounds that modulate thiamine metabolism, potentially leading to novel therapeutic strategies for neurological and metabolic disorders.

These emerging research directions highlight the continuing importance of well-characterized and validated THTPA antibodies in advancing our understanding of fundamental biological processes and disease mechanisms. As these investigations progress, they may lead to novel diagnostic markers and therapeutic approaches for conditions involving disturbed thiamine metabolism.

What are the most effective approaches for comparing and integrating data from different THTPA antibodies?

Effective comparison and integration of data from different THTPA antibodies requires systematic approaches to reconcile potential variations while maximizing reliability:

• Standardized validation matrix:

  • Develop a comprehensive validation matrix that evaluates each THTPA antibody (15486-1-AP, OTI13E3, Abbexa, HPA028876) across standardized parameters :

    • Epitope specificity

    • Detection sensitivity

    • Application performance (WB, IHC, IF, IP)

    • Species cross-reactivity

    • Lot-to-lot consistency

  • This systematic comparison establishes a foundation for integrating results across antibodies

• Cross-validation experimental design:

  • Include multiple THTPA antibodies within the same experiment when possible

  • Apply different antibodies to identical sample replicates

  • Quantify correlation between signals from different antibodies

  • Strong correlations suggest reliable detection of the same biological phenomenon

• Metadata documentation and integration:

  • Document comprehensive metadata for each experiment:

    • Antibody catalog numbers and lot numbers

    • Exact dilutions and incubation conditions

    • Detection methods and image acquisition parameters

  • This detailed documentation facilitates accurate comparison across studies and laboratories

• Statistical approaches for data integration:

  • When integrating quantitative data from different antibodies:

    • Apply normalization methods that account for antibody-specific signal characteristics

    • Use statistical models that incorporate antibody-specific variables

    • Consider meta-analysis approaches when combining results across multiple studies

• Contextual interpretation framework:

  • Develop a framework for interpreting antibody-specific findings in their proper context:

    • Polyclonal antibodies like 15486-1-AP may detect multiple THTPA isoforms

    • Monoclonal antibodies like OTI13E3 may miss certain isoforms but provide higher specificity

    • Different antibodies may have distinct performance characteristics across applications

• Repository development:

  • Establish shared data repositories that include standardized THTPA antibody validation data

  • Include raw images and quantification data to enable re-analysis

  • Link experimental results to specific antibody validation parameters