HPRT Antibody

What is HPRT and why is it significant as an antibody target in research?

HPRT (also known as HPRT1, HGPRT, or HGPRTase) is an enzyme involved in purine metabolism that converts hypoxanthine and guanine to their respective nucleotides. It serves as an excellent research target for multiple reasons:

• It is naturally expressed in growing cells and has become a model disease gene

• HPRT is drug-selectable, allowing for efficient selection systems in genetic studies

• Cells with inactive HPRT can be selected using 6-thioguanine (6TG) media

• Cells with active HPRT can be selected using hypoxanthine, aminopterin, and thymidine (HAT) media

• It has emerging roles in cancer progression and tumor immunity

This dual-selection capability makes HPRT antibodies particularly valuable for monitoring gene editing, DNA repair, and cellular transformation studies.

What experimental applications are HPRT antibodies commonly used for?

HPRT antibodies are versatile tools in molecular and cellular biology with multiple validated applications:

HPRT antibodies have been validated across human, mouse, and rat samples, allowing for comparative studies across different model systems .

How should researchers validate HPRT antibody specificity for their experiments?

Proper validation of HPRT antibodies is essential for experimental reproducibility. A comprehensive validation approach includes:

• Knockout validation: Use HPRT knockout cell lines to confirm absence of signal when the target is not present. Several commercially available antibodies have been knockout-validated specifically for HPRT .

• Multiple antibody approach: Use different antibodies targeting distinct epitopes of HPRT to confirm consistent localization patterns.

• Positive control selection: Include samples with known HPRT expression levels such as:

  • HT1080 cells (human fibrosarcoma) which have been well-characterized for HPRT expression

  • HAT-resistant cell clones with confirmed HPRT activity

• Negative control implementation: Include 6TG-resistant cells which should have inactive or absent HPRT expression .

• Peptide competition: Preincubate antibody with purified HPRT protein or peptide to demonstrate signal specificity.

This systematic approach ensures that observed signals genuinely represent HPRT rather than non-specific binding.

What are the optimal sample preparation methods for different HPRT detection techniques?

Sample preparation significantly impacts HPRT detection quality across different techniques:

For Western Blotting:

• Use RIPA or NP-40 lysis buffers supplemented with protease inhibitors

• Sonicate briefly to ensure complete nuclear protein extraction

• Include phosphatase inhibitors if studying HPRT post-translational modifications

• Load 20-40μg of total protein per lane for optimal detection

For Immunohistochemistry:

• Fixation in 10% neutral buffered formalin (24 hours maximum)

• Paraffin embedding followed by 4-5μm section cutting

• Antigen retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Primary HPRT antibody incubation at 4°C overnight using manufacturer's recommended dilution

• Visualization using PV9000 immunohistochemical detection systems

• Look for cytoplasmic staining appearing brownish-yellow or tan as positive HPRT signal

For Flow Cytometry:

• Gentle fixation with 2% paraformaldehyde

• Permeabilization with 0.1% Triton X-100 or saponin-based buffers

• Block with serum-based buffer (5-10% serum in PBS)

• Incubate with validated flow-compatible HPRT antibody clones like PAT1D9AT

How can researchers troubleshoot weak or non-specific HPRT antibody signals?

When encountering signal issues with HPRT antibodies, implement these systematic troubleshooting approaches:

For weak signals:

• Increase antibody concentration incrementally (1.5-2× manufacturer recommendation)

• Extend primary antibody incubation time (overnight at 4°C)

• Optimize antigen retrieval conditions for IHC/ICC

• Enhance detection sensitivity using amplification systems

• Enrich for HPRT-expressing cells using HAT selection

For non-specific signals:

• Increase blocking time and concentration (5% BSA or 10% serum)

• Reduce primary antibody concentration

• Include additional washing steps

• Use monoclonal antibodies like ARC1300 or PAT1D9AT for higher specificity

• Consider knockout-validated antibodies specifically tested for HPRT specificity

How can HPRT antibodies be leveraged to study DNA double-strand break repair mechanisms?

HPRT serves as an excellent platform for studying DNA repair due to its drug-selectable nature. Advanced methodologies include:

• I-SceI-mediated DNA break systems: HPRT minigenes with integrated I-SceI restriction sites allow for controlled DNA double-strand break (DSB) induction. Researchers can:

  • Generate I-SceI-sensitive human HPRT alleles in target cells

  • Transfect with I-SceI expression plasmids to induce breaks

  • Monitor repair outcomes using HAT/6TG selection systems

  • Quantify accurate non-homologous end joining (accNHEJ) frequency by measuring HAT-resistant colony formation

  • Detect mutagenic repair using 6TG resistance development

• CRISPR/Cas9 targeting: Use Cas9 nuclease to cleave HPRT alleles and evaluate repair outcomes. This approach allows:

  • Site-specific genomic targeting of endogenous HPRT

  • Comparisons between different repair template designs

  • Evaluation of homology-directed repair vs. non-homologous end joining

  • Antibody-based verification of repair outcomes

• Cell cycle-specific repair analysis: HPRT antibodies can be combined with cell cycle markers to investigate repair pathway choices across different cell cycle phases. Several I-SceI-sensitive HPRT minigenes have been specifically developed to facilitate these studies .

This methodological approach provides quantitative assessment of repair pathway usage and efficiency while avoiding confounding chromosomal position effects.

What role does HPRT play in cancer biology and how can antibodies help investigate this?

Recent research has uncovered significant roles for HPRT in cancer, with antibody-based methodologies providing important insights:

The methodological approach should include immunohistochemical staining of tumor microarrays using validated HPRT antibodies, with subsequent correlation to clinicopathological features and survival data. This provides a comprehensive view of how HPRT influences cancer progression and patient outcomes.

How can researchers employ HPRT antibodies to investigate tumor immunity interactions?

HPRT has emerging roles in tumor immunity, providing opportunities for novel immunotherapeutic approaches:

• Immune cell infiltration analysis: HPRT1 expression correlates with infiltration patterns of 22 immune cell subtypes across 33 cancer types. Researchers can:

  • Use multiplex immunofluorescence with HPRT antibodies alongside immune cell markers

  • Implement CIBERSORT computational methods to assess relative immune cell abundance

  • Apply correlation coefficient filters (cor > 0.3, P < 0.001) to identify significant associations

• Immunomodulator relationship studies: HPRT1 expression correlates with key immunoregulatory molecules:

  • Immunoinhibitors

  • Immunostimulators

  • Major histocompatibility complex (MHC) molecules

• Checkpoint inhibitor response prediction: Methodologies examining correlations between HPRT1 and PD-1/PD-L1 expression can help predict immunotherapy response, especially in triple-negative breast cancer. This approach involves:

  • Immunohistochemical scoring of PD-1/PD-L1 (0-5 scale)

  • Classification into low (0-2) versus high (3-5) expression groups

  • Correlation analysis with HPRT1 expression levels

This integrated approach allows researchers to explore HPRT's role in modulating the immunosuppressive tumor microenvironment, potentially improving cancer immunotherapy outcomes.

What are the methodological considerations for studying HPRT in gene editing and genetic engineering experiments?

HPRT provides an excellent selection system for gene editing experiments due to its dual-selection capability:

• Reporter system design: Creating HPRT minigenes containing I-SceI recognition sites:

  • Clone HPRT minigenes into appropriate expression vectors

  • Integrate I-SceI sites at strategic locations

  • Establish stable cell lines containing the modified minigenes

  • Verify integration using HPRT antibodies and Southern blot analysis

• Selection system implementation:

  • 6TG selection identifies cells with inactive HPRT (survival indicates gene disruption)

  • HAT selection identifies cells with functional HPRT (survival indicates successful correction)

  • Combined approach allows bidirectional selection for gene editing events

• Quantification methodologies:

  • Colony formation assays to determine editing efficiencies

  • Flow cytometry using HPRT antibodies to assess population-level changes

  • Single-cell isolation and expansion to establish edited clones

• Verification strategies:

  • Southern blot analysis to confirm genomic modifications

  • PCR amplification and sequencing of targeted regions

  • Western blot using HPRT antibodies to verify protein expression changes

This systematic approach enables precise quantification of gene editing efficiency while avoiding confounding chromosomal position effects that can complicate interpretation.

How can HPRT antibodies help investigate post-translational modifications and protein interactions?

Advanced protein interaction studies involving HPRT require specialized antibody-based techniques:

• Immunoprecipitation protocols:

  • Use highly specific monoclonal antibodies (such as clone ARC1300)

  • Implement crosslinking strategies to capture transient interactions

  • Couple with mass spectrometry for unbiased interactome analysis

• Post-translational modification (PTM) mapping:

  • Combine PTM-specific antibodies with HPRT pulldown approaches

  • Implement phospho-specific, acetylation, or ubiquitination detection methods

  • Correlate modifications with functional changes in HPRT activity

• Proximity ligation assays (PLA):

  • Use paired antibody approach (HPRT + putative interacting partner)

  • Visualize interactions at subcellular resolution

  • Quantify interaction frequency changes under different conditions

• Chromatin association studies:

  • Implement chromatin immunoprecipitation (ChIP) using HPRT antibodies

  • Identify potential genomic binding sites and transcriptional roles

  • Correlate with p53 regulation, as p53 significantly affects HPRT1 expression

These methodological approaches expand our understanding of HPRT beyond its enzymatic function, revealing its roles in signaling networks and transcriptional regulation that may contribute to its emerging importance in cancer biology.