HPT1 Antibody

What is the HPRT1 antibody and what is its primary research application?

HPRT1 (hypoxanthine phosphoribosyltransferase 1) antibody is a research reagent that specifically targets the HPRT1 protein, which plays a central role in the generation of purine nucleotides through the purine salvage pathway . The antibody is primarily used in various immunological detection methods including Western Blot (WB), immunohistochemistry (IHC), immunofluorescence (IF/ICC), flow cytometry (FC), and immunoprecipitation (IP) . Research applications focus on purine metabolism studies, genetic disorder investigations related to HPRT1 mutations, and as a reference gene marker in expression studies.

What species reactivity has been confirmed for HPRT1 antibody?

HPRT1 antibody has demonstrated confirmed reactivity with human, mouse, and rat samples in multiple experimental applications . Western blot analysis has specifically verified positive detection in human cell lines (HeLa, HEK-293, A549, MCF-7, HepG2, Jurkat, and Y79), mouse tissues (liver and brain), and rat brain tissue . This multi-species reactivity makes the antibody particularly valuable for comparative studies and translational research between model organisms and human systems.

What are the recommended dilutions for different experimental applications?

The following table outlines the optimal dilution ranges for various experimental applications:

These dilutions should be optimized for each specific experimental system to obtain reliable results . The observed molecular weight of HPRT1 is typically between 24-28 kDa, which aligns with its calculated molecular weight of 25 kDa .

What is the difference between monoclonal and polyclonal antibodies for research applications?

In research applications, the choice between monoclonal and polyclonal antibodies depends on experimental goals. Polyclonal antibodies like the rabbit anti-HPRT1 antibody recognize multiple epitopes on the target antigen, providing stronger signals but potentially more background . In contrast, monoclonal antibodies (such as those generated against HPT) recognize a single epitope, offering higher specificity but sometimes lower sensitivity .

For instance, the anti-HPT monoclonal antibodies (F1, D4-2, D4-4, and D4-5) demonstrated high specificity with ascites ELISA titers ranging from 1:10,000 to 1:100,000, making them suitable for precise detection applications . The choice depends on whether experimental priorities are signal strength and robustness (polyclonal) or epitope-specific precision (monoclonal).

How should samples be prepared for optimal HPRT1 antibody detection?

For optimal HPRT1 antibody detection, sample preparation varies by application. For Western blot analysis, total protein extraction using RIPA buffer with protease inhibitors is recommended, followed by quantification and denaturation in Laemmli buffer with β-mercaptoethanol . For immunohistochemistry, antigen retrieval using TE buffer (pH 9.0) has been demonstrated to yield optimal results, though citrate buffer (pH 6.0) may serve as an alternative .

For immunofluorescence applications, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 provides consistent results. For flow cytometry, intracellular staining requires fixation and permeabilization to allow antibody access to the intracellular HPRT1 protein . In all applications, inclusion of appropriate positive controls (such as HeLa cells) and negative controls (isotype or pre-immunization serum) is essential for result validation.

What methodologies are effective for generating new antibodies against related targets?

For generating new antibodies against related targets, hybridoma technology has proven effective as demonstrated in the development of anti-HPT monoclonal antibodies . This approach involves:

• Immunization of BALB/c mice with purified recombinant protein (e.g., 6His-tagged target protein)

• Isolation of spleen cells and fusion with myeloma cells

• Selection of hybrid cells through culture in HAT medium

• Screening for antibody-producing clones via ELISA

• Cloning and expansion of positive hybridomas

• Characterization of produced antibodies for specificity, titer, and isotype

For human antibodies, an alternative approach involves isolating specific B-cells from immunized individuals using fluorescently labeled antigens, followed by stimulation of antibody production in culture, and subsequent cloning of variable regions to generate recombinant monoclonal antibodies . This method was successfully used to isolate HPA-1a-specific B-cells and generate monoclonal antibodies from maternal serum .

How can researchers assess antibody specificity and minimize cross-reactivity?

Assessing antibody specificity and minimizing cross-reactivity requires multiple validation approaches:

• Western blot analysis: Test the antibody against various cell/tissue lysates to confirm target-specific binding at the expected molecular weight (24-28 kDa for HPRT1)

• Knockout/knockdown validation: Compare antibody reactivity between wild-type samples and those with the target protein depleted (genetic knockout or siRNA knockdown)

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide or recombinant protein to confirm specific binding is blocked

• Multiple application testing: Verify consistent target recognition across different applications (WB, IHC, IF/ICC) to ensure epitope-specific binding

• Cross-species reactivity testing: Evaluate antibody performance across species (human, mouse, rat) to confirm evolutionary conservation of the recognized epitope

For HPRT1 antibody specifically, published validation data from 31 Western blot studies, 2 IHC applications, and 2 IF applications provide substantial evidence for specificity .

How can HPRT1 antibody be applied in studies of purine metabolism disorders?

HPRT1 antibody serves as a valuable tool in studying purine metabolism disorders, particularly Lesch-Nyhan syndrome and related conditions characterized by HPRT1 dysfunction. Advanced research applications include:

• Protein-level verification of genetic mutations: Using HPRT1 antibody in Western blot analysis to detect altered protein expression, truncation, or complete absence in patient-derived samples

• Intracellular localization studies: Employing immunofluorescence with HPRT1 antibody to investigate potential aberrant subcellular localization of mutant proteins

• Protein-protein interaction analysis: Utilizing immunoprecipitation with HPRT1 antibody to identify altered interaction partners in disease states

• Tissue-specific expression patterns: Applying immunohistochemistry to characterize differential expression across tissues in normal versus pathological conditions

These approaches can provide critical insights into disease mechanisms beyond genomic analysis, helping to establish genotype-phenotype correlations in purine metabolism disorders.

What are the considerations for multiplexing HPRT1 antibody with other markers?

When multiplexing HPRT1 antibody with other markers for co-localization or comparative expression studies, several technical considerations are essential:

• Host species compatibility: Since the HPRT1 antibody is rabbit-derived (IgG) , secondary antibodies should be chosen to avoid cross-reactivity. For multiplexing, pair with primary antibodies from different host species (mouse, goat, chicken)

• Spectral separation: When using fluorescent detection, select fluorophores with minimal spectral overlap to ensure clear discrimination between signals

• Antibody validation in multiplex conditions: Validate each antibody individually before combining to establish baseline signals and confirm no unexpected interference

• Sequential versus simultaneous staining: For challenging combinations, sequential staining protocols with blocking steps between applications can minimize cross-reactivity

• Controls for multiplexing: Include single-stained controls alongside multiplexed samples to verify specific signal detection

This approach is particularly valuable for studying HPRT1 in relation to other components of the purine salvage pathway or when using HPRT1 as a housekeeping gene control alongside proteins of interest.

How can researchers troubleshoot non-specific binding issues with HPRT1 antibody?

When encountering non-specific binding with HPRT1 antibody, several troubleshooting strategies can be implemented:

• Optimize antibody concentration: Further titration might be necessary from the recommended ranges (1:2000-1:10000 for WB, 1:20-1:200 for IHC)

• Increase blocking stringency: Use 5% BSA or 5% non-fat dry milk in TBS-T with extended blocking times (2+ hours at room temperature)

• Modify washing protocols: Implement additional and longer washing steps with increased detergent concentration (0.1-0.5% Tween-20)

• Adjust antigen retrieval parameters: For IHC applications, compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) antigen retrieval methods

• Evaluate buffer compatibility: Ensure the antibody storage buffer (PBS with 0.02% sodium azide and 50% glycerol) is compatible with the experimental system

• Consider pre-adsorption: For tissues with high background, pre-adsorb the antibody with tissue powder from the species being tested

These approaches systematically address the most common sources of non-specific binding while preserving specific target recognition.

How does the performance of polyclonal HPRT1 antibody compare to monoclonal alternatives?

The polyclonal HPRT1 antibody offers distinct advantages and limitations compared to monoclonal alternatives:

The rabbit polyclonal anti-HPRT1 antibody has demonstrated broad application versatility with validated performance in multiple techniques and across species barriers , making it suitable for diverse research applications, while monoclonal alternatives might offer advantages in highly specific detection scenarios.

What are the emerging applications for HPRT1 antibody in cancer research?

HPRT1 antibody is finding emerging applications in cancer research beyond its traditional role as a housekeeping gene control:

• Metabolic reprogramming studies: As cancer cells often exhibit altered purine metabolism, HPRT1 antibody can help characterize changes in the purine salvage pathway activity across different cancer types

• Therapy resistance mechanisms: Changes in HPRT1 expression may correlate with resistance to certain chemotherapeutic agents that target nucleotide synthesis

• Cancer stem cell identification: Combined with other markers, HPRT1 expression patterns may help identify cancer stem cell populations with distinct metabolic profiles

• Prognostic biomarker development: Correlation of HPRT1 expression levels with patient outcomes across cancer types may reveal prognostic value

• Therapeutic target validation: For cancers dependent on purine salvage pathway activity, HPRT1 antibody can help validate this pathway as a potential therapeutic target

When employing HPRT1 antibody in cancer research, validation in relevant human cancer cell lines has been extensively documented, including HeLa, MCF-7, A549, HepG2, and Jurkat cells .

How can HPRT1 antibody be utilized in enzyme activity correlation studies?

HPRT1 antibody can be strategically employed in enzyme activity correlation studies to establish relationships between protein expression and functional activity:

• Paired protein-activity analysis: Researchers can divide samples for parallel HPRT1 protein quantification (via Western blot) and enzymatic activity assays (spectrophotometric)

• Immunocapture enzyme activity assays: Immobilize HPRT1 antibody on plates or beads to capture the enzyme from complex samples, then directly measure activity of the captured protein

• Expression-activity correlation in mutant studies: Compare HPRT1 protein levels with enzyme activity across wild-type and mutant variants to establish structure-function relationships

• Inhibitor screening validation: Confirm that reduced HPRT1 activity in inhibitor screens corresponds to maintained protein levels rather than protein degradation

• Post-translational modification impact: Investigate how specific modifications (detectable through other antibodies) correlate with changes in enzyme activity

This integrated approach provides deeper insight into regulatory mechanisms affecting HPRT1 function beyond simple presence or absence of the protein, potentially revealing novel therapeutic intervention points.

What protocols can enhance sensitivity when working with low-abundance HPRT1 in specific tissues?

When investigating low-abundance HPRT1 expression in specific tissues, several protocol enhancements can improve detection sensitivity:

• Signal amplification systems: Employ tyramide signal amplification (TSA) or other enzymatic amplification methods to enhance detection in IHC and IF applications

• Sample enrichment strategies: Use subcellular fractionation to concentrate cytosolic fractions where HPRT1 is predominantly located

• Extended antibody incubation: Increase primary antibody incubation time to 48-72 hours at 4°C with gentle agitation to improve antigen binding

• Detection system optimization: For Western blot, use high-sensitivity ECL substrates or fluorescent secondary antibodies with digital imaging

• Antigen retrieval optimization: For tissues with dense extracellular matrix, extend antigen retrieval time or utilize pressure-assisted retrieval methods

• Tissue-specific blocking: Incorporate specific blocking agents based on tissue type (mouse tissues may benefit from mouse-on-mouse blocking reagents)

When applying these enhancements, it remains essential to maintain appropriate controls to distinguish true signal from amplified background.

How should researchers validate knockout models using HPRT1 antibody?

Comprehensive validation of HPRT1 knockout models requires a multi-faceted approach:

• Multiple epitope targeting: Use antibodies targeting different HPRT1 epitopes to confirm complete protein absence rather than truncation

• Dilution series analysis: Perform Western blot with a dilution series of wild-type control to establish detection limits and confirm true absence versus below-detection expression

• Tissue-specific validation: Examine HPRT1 expression across multiple tissues, as some knockout models may retain tissue-specific expression

• Functional confirmation: Complement antibody-based detection with enzymatic activity assays to confirm functional knockout

• Specificity controls: Include known positive controls (HeLa cells, brain tissue) alongside knockout samples in all experiments

• Genomic verification: Correlate antibody results with genomic analysis (sequencing) to confirm the genetic modification produced the expected protein change

This comprehensive approach ensures knockout model validity and can identify unexpected compensatory mechanisms or splice variants that might complicate interpretation.

What are the emerging technologies for next-generation antibody development against metabolic enzymes?

Next-generation antibody development technologies for metabolic enzymes like HPRT1 are advancing rapidly:

• Recombinant antibody engineering: Use of phage display and synthetic libraries to generate highly specific antibodies with reduced batch variation compared to traditional hybridoma methods

• Nanobody development: Generation of single-domain antibodies with superior tissue penetration and recognition of cryptic epitopes in metabolic enzymes

• Conformation-specific antibodies: Development of antibodies that specifically recognize active versus inactive enzyme conformations to directly assess functional status

• Humanized antibody production: Creation of humanized versions of research antibodies to facilitate translational applications and potential therapeutic development

• Site-specific conjugation strategies: Advancement of conjugation chemistries that maintain binding characteristics while adding detection or enrichment capabilities

These technologies promise to enhance specificity, reproducibility, and functional insight in metabolic enzyme research, potentially revealing new roles for HPRT1 in health and disease.

How might standardized antibody validation criteria improve reproducibility in HPRT1 research?

Implementing standardized antibody validation criteria would significantly enhance reproducibility in HPRT1 research:

• Application-specific validation: Require demonstration of specificity in each claimed application (WB, IHC, IF/ICC, FC, IP) rather than extrapolating from a single technique

• Genetic approach integration: Mandate validation using genetic models (knockout/knockdown) as the gold standard for specificity

• Independent validation requirements: Establish minimum standards for independent laboratory confirmation before antibody commercialization

• Epitope mapping documentation: Require detailed epitope information to predict potential cross-reactivity and sensitivity to protein modifications

• Quantitative performance metrics: Develop standardized sensitivity, specificity, and reproducibility metrics across applications and sample types

• Transparent reporting standards: Implement comprehensive reporting of validation methods, positive/negative controls, and batch information in publications