HPRT1 Antibody

What is HPRT1 and why is it an important research target?

HPRT1 (also known as hypoxanthine-guanine phosphoribosyltransferase or HGPRTase) is a transferase enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate via transfer of the 5-phosphoribosyl group from 5-phosphoribosyl 1-pyrophosphate. This enzyme plays a central role in the generation of purine nucleotides through the purine salvage pathway . Beyond its metabolic functions, HPRT1 has emerged as an important research target due to its involvement in several pathological conditions. Mutations in HPRT1 result in Lesch-Nyhan syndrome (LNS) or gout . Additionally, recent research indicates HPRT1 is overexpressed in various cancers including nasopharyngeal carcinoma, breast cancer, endometrial cancer, lung cancer, and prostate cancer, where it's associated with poor prognosis . This dual role in metabolic disorders and cancer makes HPRT1 a compelling research target.

What types of HPRT1 antibodies are available for research?

Researchers have access to both monoclonal and polyclonal HPRT1 antibodies from various sources, with different host species and target epitopes:

The choice between these options depends on the specific experimental requirements and applications. Monoclonal antibodies offer high specificity to a single epitope, while polyclonals may provide higher sensitivity by binding multiple epitopes .

What applications are HPRT1 antibodies validated for in research?

HPRT1 antibodies have been validated for multiple research applications, as documented across various suppliers and publications:

The versatility of these applications allows researchers to study HPRT1 expression, localization, interactions, and modifications in various experimental contexts .

How do I select the appropriate HPRT1 antibody for my research?

When selecting an HPRT1 antibody, consider these methodological factors:

• Experimental application: Different antibodies perform optimally in specific applications. For example, some HPRT1 antibodies are specifically validated for Western blot but may not work well for IHC .

• Species reactivity: Confirm the antibody recognizes HPRT1 from your species of interest. Many HPRT1 antibodies react with human, mouse, and rat samples, but cross-reactivity should be verified .

• Antibody type:

  • Monoclonal antibodies (e.g., clone 5F11A7) offer high specificity and reproducibility, ideal for quantitative analyses

  • Polyclonal antibodies provide higher sensitivity by recognizing multiple epitopes, beneficial for detection of low-abundance targets

• Validation data: Review the antibody's validation data, including Western blot images showing the expected 24-28 kDa band and positive controls in relevant tissues .

• Epitope location: Consider whether your research requires targeting a specific region of HPRT1. Some antibodies target full-length protein (AA 1-218), while others target specific domains .

• KO/KD validation: For critical research, select antibodies validated in knockout/knockdown systems to ensure specificity .

The optimal choice depends on your specific experimental goals, target species, and detection system.

What are the molecular characteristics of HPRT1 that influence antibody selection?

Understanding HPRT1's molecular characteristics is essential for effective antibody selection:

• Molecular weight: HPRT1 has a calculated molecular weight of approximately 25 kDa, with observed molecular weight in Western blot typically between 24-28 kDa . This information is crucial for verifying antibody specificity.

• Cellular localization: HPRT1 is predominantly cytoplasmic , so antibodies for immunofluorescence should effectively detect cytoplasmic signals.

• Sequence conservation: HPRT1 is highly conserved across species, explaining why many antibodies show cross-reactivity between human, mouse, and rat samples .

• Important domains: The protein contains catalytic domains involved in substrate binding and enzyme activity. Antibodies targeting different regions may have different effects on protein function in experimental settings .

• Post-translational modifications: Consider whether your research questions involve detecting modified forms of HPRT1, as some antibodies may have differential recognition of phosphorylated or other modified forms .

• Expression levels: HPRT1 expression varies across tissues and is notably elevated in certain cancer types, potentially requiring antibodies with different sensitivity ranges depending on your experimental system .

These molecular characteristics should guide antibody selection to ensure optimal detection and specificity for your particular research focus.

What are the recommended protocols for using HPRT1 antibodies in Western blot?

For optimal Western blot results with HPRT1 antibodies, follow these methodological guidelines:

• Sample preparation:

  • Use cell lysates from validated positive controls (HeLa, HEK-293, NIH/3T3)

  • Load 20-30 μg of total protein per lane

  • Include appropriate positive controls (e.g., recombinant HPRT1 protein)

• Electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 24-28 kDa

  • Transfer proteins to PVDF or nitrocellulose membranes using standard methods

• Antibody dilutions and incubation:

  • Primary antibody: Use at 1:1000-1:5000 dilution for monoclonal antibodies or 1:2000-1:10000 for polyclonal antibodies

  • Incubate with primary antibody overnight at 4°C or for 2 hours at room temperature

  • Secondary antibody: Choose appropriate HRP-conjugated antibody at 1:5000-1:10000 dilution

• Detection and analysis:

  • Develop using ECL or similar detection reagents

  • Expect a band at approximately 24-28 kDa for HPRT1

  • For quantitative analysis, normalize to appropriate loading controls (e.g., GAPDH, β-actin)

Studies have shown that HPRT1 protein levels are significantly elevated in cancer cells compared to normal controls, with approximately 47% of cancer patients showing upregulation . This should be considered when interpreting Western blot results from clinical samples.

How should I optimize immunohistochemistry protocols for HPRT1 detection?

For successful immunohistochemical detection of HPRT1 in tissue sections:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) sections (4-6 μm thickness)

  • Include positive control tissues (e.g., human liver, colon cancer, endometrial cancer)

• Antigen retrieval (critical step):

  • Recommended: TE buffer pH 9.0 for optimal results

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval using pressure cooker or microwave

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • Primary antibody dilutions:

    • Monoclonal: 1:4000-1:16000

    • Polyclonal: 1:20-1:200

  • Incubate at 4°C overnight or 1-2 hours at room temperature

• Detection systems:

  • Use polymer-based detection systems (e.g., PV9000 immunohistochemical detection kit)

  • DAB chromogen for visualization

  • Counterstain with hematoxylin

• Scoring and interpretation:

  • Follow established scoring methods:

    • Staining intensity: 0 (no staining), 1 (light), 2 (moderate), 3 (heavy)

    • Percentage of positive cells: 0 (none), 1 (1-25%), 2 (26-50%), 3 (>50%)

    • Combined score: 0-2 (negative), 3-4 (positive +), 5-6 (positive ++)

Research has demonstrated that HPRT1 staining patterns differ between normal and cancer tissues, with increased intensity and cytoplasmic localization in tumors, particularly in nasopharyngeal carcinoma and other cancers .

What protocols yield optimal results for immunofluorescence with HPRT1 antibodies?

For high-quality immunofluorescence detection of HPRT1:

• Cell preparation:

  • Culture cells on coverslips or chamber slides to 70-80% confluence

  • Validated cell lines include HeLa, NIH/3T3, and various cancer cell lines

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

  • Alternative: Fix and permeabilize simultaneously with methanol at -20°C for 10 minutes

• Blocking and antibody incubation:

  • Block with 3-5% BSA or normal serum for 30-60 minutes

  • Primary antibody dilutions:

    • Polyclonal: 1:200-1:800

    • Monoclonal: 1:400-1:1600

  • Incubate 1-2 hours at room temperature or overnight at 4°C

• Secondary antibody and nuclear counterstaining:

  • Use fluorophore-conjugated secondary antibodies appropriate for your microscopy setup

  • Counterstain nuclei with DAPI or Hoechst

  • Mount with anti-fade mounting medium

• Imaging and analysis:

  • HPRT1 typically shows cytoplasmic localization

  • For co-localization studies, select compatible fluorophores for multi-channel imaging

  • Use appropriate positive and negative controls for accurate interpretation

Research has shown that HPRT1 expression patterns can vary between normal and cancer cells, with potential diagnostic and prognostic implications . For quantitative analysis, standardized image acquisition and analysis parameters should be established.

How can I use HPRT1 antibodies to study protein-protein interactions?

To investigate HPRT1 protein interactions, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use 0.5-4.0 μg of HPRT1 antibody per 1-3 mg of total protein lysate

  • Optimize lysis buffers to preserve protein interactions (typically RIPA or NP-40 based)

  • Validate interactions by reverse Co-IP using antibodies against suspected interacting partners

  • Western blot analysis of immunoprecipitates to confirm interactions

• Proximity Ligation Assay (PLA):

  • Use dilutions of 1:400-1:800 of HPRT1 antibody

  • Combine with antibodies against suspected interaction partners

  • Visualize interactions as fluorescent dots representing proteins within 40 nm proximity

  • Quantify signal to assess interaction strength

• Immunofluorescence co-localization:

  • Use HPRT1 antibody dilutions of 1:200-1:800

  • Combine with antibodies against potential interacting proteins

  • Analyze co-localization using appropriate software (e.g., ImageJ with co-localization plugins)

  • Calculate Pearson's or Mander's coefficients to quantify co-localization

• Pull-down assays with recombinant proteins:

  • Use recombinant HPRT1 as bait

  • Confirm interactions using HPRT1 antibodies in Western blot

  • Identify novel interacting partners by mass spectrometry

Recent research suggests potential interactions between HPRT1 and proteins involved in cancer-related pathways, including CyclinD1, CyclinE, MDR1, MMP-2, and MMP-9 . These interactions may contribute to HPRT1's role in cancer progression and chemoresistance, making them important targets for investigation.

What are the optimal storage and handling conditions for HPRT1 antibodies?

To maintain optimal activity and specificity of HPRT1 antibodies, follow these storage and handling guidelines:

• Storage temperature:

  • Store at -20°C for long-term storage

  • Aliquoting is generally unnecessary for -20°C storage, as indicated by manufacturers

  • Avoid repeated freeze-thaw cycles

• Buffer composition:

  • Most HPRT1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Some formulations may include BSA as a stabilizer (e.g., 0.1% BSA in smaller size aliquots)

• Working dilutions and temporary storage:

  • Prepare working dilutions immediately before use

  • Working dilutions can typically be stored at 4°C for up to one week

  • For longer storage of working dilutions, add stabilizing proteins (e.g., 0.1-1% BSA)

• Antibody stability:

  • Most manufacturers indicate stability for one year after shipment when stored properly

  • Monitor for signs of degradation such as precipitation, clouding, or loss of activity

• Shipping and temporary storage:

  • HPRT1 antibodies are typically shipped with ice packs

  • Upon receipt, transfer immediately to -20°C for long-term storage

  • Brief storage at 4°C is acceptable for antibodies in glycerol buffer but should be minimized

Proper storage and handling ensure consistent antibody performance across experiments, which is particularly important for quantitative studies examining HPRT1 expression in cancer research and other applications .

Why might I see multiple bands when using HPRT1 antibodies in Western blot?

Multiple bands in HPRT1 Western blots may occur for several reasons, requiring different troubleshooting approaches:

• Post-translational modifications:

  • HPRT1 may undergo phosphorylation or other modifications resulting in shifted bands

  • Compare with literature to identify if additional bands correspond to known modified forms

  • Use phosphatase treatment of samples to determine if additional bands are phosphorylated forms

• Proteolytic degradation:

  • Add protease inhibitors to lysis buffer (complete protease inhibitor cocktail)

  • Keep samples cold during preparation

  • Reduce sample handling time before adding SDS sample buffer

  • Look for lower molecular weight bands that may represent degradation products

• Non-specific binding:

  • Increase antibody dilution (try 1:5000 instead of 1:1000)

  • Optimize blocking conditions (5% non-fat milk or BSA)

  • Increase washing duration and number of washes

  • Consider using a more specific monoclonal antibody like clone 5F11A7

• Splice variants:

  • Compare band patterns with known splice variant molecular weights

  • Consult literature or databases for HPRT1 splice variants

  • Validate with RT-PCR or other methods if splice variants are suspected

• Cross-reactivity:

  • Test the antibody on knockout or knockdown samples if available

  • Compare results with a different HPRT1 antibody targeting a different epitope

  • Perform peptide competition assays to confirm specificity

The expected molecular weight for HPRT1 is 24-28 kDa . Any additional bands should be carefully evaluated against these potential causes.

How can I improve signal-to-noise ratio in HPRT1 immunohistochemistry?

To enhance signal-to-noise ratio in HPRT1 immunohistochemistry:

• Optimize antigen retrieval:

  • Compare TE buffer pH 9.0 (recommended) with citrate buffer pH 6.0

  • Test different retrieval durations (10-30 minutes)

  • Optimize retrieval temperature and cooling periods

• Titrate primary antibody:

  • Test a range of dilutions; recommended ranges:

    • Monoclonal: 1:4000-1:16000

    • Polyclonal: 1:20-1:200

  • Higher dilutions often reduce background while maintaining specific signal

• Optimize blocking conditions:

  • Increase blocking duration (1-2 hours)

  • Test different blocking agents (normal serum, BSA, commercial blockers)

  • Consider dual blocking with serum followed by protein block

• Reduce non-specific binding:

  • Add 0.1-0.3% Triton X-100 to antibody diluent

  • Increase washing steps (5-6 washes of 5 minutes each)

  • Use TBS-T instead of PBS-T if high background persists

• Detection system considerations:

  • Use polymer-based detection systems for improved signal-to-noise ratio

  • Optimize DAB development time (monitor under microscope)

  • Consider amplification systems for weak signals

• Tissue-specific considerations:

  • For tissues with high endogenous peroxidase, increase H₂O₂ quenching time

  • For high-biotin tissues, use biotin blocking kits before antibody incubation

  • For tissues with high background, consider adding 5% non-fat milk to antibody diluent

In research studying HPRT1 in cancer tissues, researchers have successfully used these optimization steps to achieve clear distinction between HPRT1-positive and negative samples, enabling accurate scoring and classification .

What are the most common causes of false positives/negatives in HPRT1 detection?

Understanding potential sources of false results is critical for accurate HPRT1 detection:

Common causes of false positives:

• Cross-reactivity with similar proteins:

  • Validate antibody specificity using knockout/knockdown controls

  • Use monoclonal antibodies with well-characterized epitopes (e.g., clone 5F11A7)

  • Confirm results with a second antibody targeting a different epitope

• Endogenous enzyme activity:

  • In IHC: Ensure complete quenching of endogenous peroxidase (3% H₂O₂, 10-15 minutes)

  • In IF: Use appropriate controls to distinguish autofluorescence from specific signal

• Non-specific binding of secondary antibodies:

  • Include secondary-only controls

  • Use secondary antibodies pre-adsorbed against species in your samples

  • Optimize secondary antibody dilution (typically 1:10000-1:15000)

Common causes of false negatives:

• Inadequate antigen retrieval:

  • HPRT1 detection in FFPE tissues requires effective antigen retrieval

  • Optimize pH (recommended: TE buffer pH 9.0)

  • Ensure adequate temperature and duration

• Antibody degradation:

  • Store antibodies according to manufacturer recommendations (-20°C)

  • Avoid repeated freeze-thaw cycles

  • Check expiration dates and storage conditions

• Suboptimal protein extraction:

  • For Western blot: Use appropriate lysis buffers (RIPA or NP-40 based)

  • Include protease inhibitors to prevent degradation

  • Optimize protein extraction protocol for your specific sample type

• Epitope masking or alteration:

  • Consider issues with fixation duration in tissue samples

  • Test multiple antibodies targeting different epitopes

  • For modified forms, use specific antibodies designed to detect phosphorylated or other modified forms

How should I validate HPRT1 antibody specificity for my research?

Rigorous validation of HPRT1 antibody specificity is essential for robust research findings:

• Genetic validation approaches:

  • Test on HPRT1 knockout or knockdown samples compared to wild-type controls

  • Use siRNA-mediated knockdown (e.g., validated HPRT1-siRNA3 has shown high knockdown efficiency)

  • Perform antibody testing on overexpression systems

• Biochemical validation methods:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide before application

  • Test for the expected 24-28 kDa band in Western blot

  • Compare results across multiple antibodies targeting different HPRT1 epitopes

• Positive and negative controls:

  • Include validated positive controls (e.g., HeLa, HEK-293, Jurkat, NIH/3T3 cells)

  • Use tissues known to express HPRT1 (e.g., liver, brain)

  • Include negative controls (secondary antibody only, isotype controls)

• Cross-platform validation:

  • Confirm findings across multiple techniques (e.g., WB, IHC, IF)

  • Compare protein detection with mRNA expression data

  • Correlate with functional assays where possible

• Literature comparison:

  • Compare your results with published data on HPRT1 expression patterns

  • Reference the molecular weight and localization patterns reported in literature

  • Note any discrepancies and investigate potential causes

Research has shown that HPRT1 knockdown leads to decreased expression of specific proteins including CyclinD1, CyclinE, MDR1, MMP-2, and MMP-9 . Validation studies should include assessment of these known downstream effects to confirm antibody specificity and biological relevance.

What control samples should I include when working with HPRT1 antibodies?

A comprehensive set of controls ensures reliable interpretation of HPRT1 antibody experiments:

Positive controls:

• Cell lines with confirmed HPRT1 expression:

  • Human: HeLa, HEK-293, Jurkat, K-562, MCF-7, A549

  • Mouse: NIH/3T3, mouse brain tissue, mouse liver tissue

  • Rat: HSC-T6, rat brain tissue, rat heart tissue

• Tissue samples with known HPRT1 expression:

  • Human: liver, colon, brain, pancreas

  • Cancer tissues: liver cancer, colon cancer, endometrial cancer

• Recombinant protein controls:

  • Purified recombinant HPRT1 protein

  • Cells transfected with HPRT1 expression constructs

Negative controls:

• Technical negative controls:

  • Secondary antibody only (omit primary antibody)

  • Isotype control antibody (matched isotype, irrelevant specificity)

  • Pre-immune serum (for polyclonal antibodies)

• Biological negative controls:

  • HPRT1 knockout or knockdown samples

  • Tissues known to have low HPRT1 expression

  • Competition with immunizing peptide/antigen

Experimental validation controls:

• Loading controls for Western blot:

  • Housekeeping proteins (β-actin, GAPDH, tubulin)

  • Total protein staining (Ponceau S, SYPRO Ruby)

• Staining controls for IHC/IF:

  • Adjacent normal tissue in cancer studies

  • Nuclear counterstain (hematoxylin, DAPI) to assess tissue/cell morphology

  • Known marker proteins for co-localization studies

Including appropriate controls is particularly important in cancer research, where HPRT1 expression differences between normal and malignant tissues are being evaluated for diagnostic and prognostic purposes .

How can HPRT1 antibodies be used to study its role in cancer progression?

HPRT1 antibodies have become valuable tools for investigating its emerging role in cancer progression:

• Expression analysis in clinical samples:

  • IHC analysis of tumor microarrays (TMAs) to correlate HPRT1 expression with:

    • Tumor stage and grade

    • Patient survival outcomes

    • Metastatic potential

  • Western blot quantification comparing tumor vs. adjacent normal tissues

  • Research has demonstrated HPRT1 overexpression in multiple cancer types, including nasopharyngeal carcinoma, where high expression correlates with poor prognosis

• Functional studies using knockout/knockdown approaches:

  • Validate knockdown efficiency using HPRT1 antibodies in Western blot

  • Examine phenotypic changes following HPRT1 silencing:

    • Decreased cell viability and proliferation (quantified by MTT assay)

    • Reduced colony formation

    • Inhibited migration (wound healing assay)

    • Diminished invasion (transwell assay)

• Mechanistic investigations:

  • Analyze expression of downstream targets after HPRT1 manipulation:

    • Cell cycle regulators (CyclinD1, CyclinE)

    • Drug resistance mediators (MDR1)

    • Metastasis promoters (MMP-2, MMP-9)

  • Study signaling pathway alterations:

    • MMP1/PI3K/AKT pathway activation

    • Potential regulation by miRNAs (e.g., miRNA-125b-5p, miRNA-199a-5p)

• Tumor microenvironment interactions:

  • Correlate HPRT1 expression with immune cell infiltration

  • Examine relationship with StromalScore, ImmuneScore, and TumorPurity

  • Analyze associations with markers of:

    • Immunoinhibitors

    • Immunostimulators

    • MHC molecules

These approaches have revealed that HPRT1 silencing significantly reduces cancer cell proliferation and metastatic potential, suggesting its potential as a therapeutic target .

What approaches can be used to study HPRT1's involvement in chemoresistance?

Investigating HPRT1's role in chemoresistance requires specialized methodological approaches:

• Expression analysis in resistant vs. sensitive cells:

  • Western blot quantification of HPRT1 in:

    • Parental cancer cell lines

    • Chemoresistant derivatives

    • Patient samples (responders vs. non-responders)

  • Recommended antibody dilutions: 1:1000-1:5000 for monoclonal or 1:2000-1:10000 for polyclonal antibodies

• Manipulation of HPRT1 expression:

  • Knockdown with validated siRNAs (e.g., HPRT1-siRNA3)

  • Overexpression using expression vectors

  • Monitor changes in drug sensitivity (IC50 values) using:

    • MTT/MTS assays

    • Colony formation assays

    • Apoptosis assays (Annexin V/PI staining)

• Mechanistic studies:

  • Analyze expression of known chemoresistance mediators after HPRT1 manipulation:

    • MDR1 (P-glycoprotein) expression

    • ABC transporters

    • Anti-apoptotic proteins

  • Investigate drug accumulation/efflux in HPRT1-modified cells

  • Examine involvement in DNA damage repair pathways

• Signaling pathway analysis:

  • Assess PI3K/AKT pathway activation status using phospho-specific antibodies

  • Evaluate MMP expression and activity (gelatin zymography)

  • Investigate potential connections to:

    • NF-κB signaling

    • MAPK pathways

    • Autophagy induction

• Combination treatment approaches:

  • Test synergy between HPRT1 inhibition and chemotherapeutic agents

  • Evaluate combination indices to quantify synergistic effects

  • Monitor biomarkers of response using HPRT1 antibodies

Research has shown that HPRT1 knockdown significantly reduces MDR1 expression in nasopharyngeal carcinoma cells, suggesting a potential mechanism for its involvement in chemoresistance . This finding opens avenues for developing strategies to overcome treatment resistance in cancer patients.

How can I investigate HPRT1's relationship with tumor immune microenvironment?

Exploring the connections between HPRT1 and the tumor immune microenvironment requires sophisticated analytical approaches:

• Multiplex immunohistochemistry/immunofluorescence:

  • Combine HPRT1 antibodies (1:20-1:16000 dilution) with markers for:

    • T cells (CD3, CD4, CD8)

    • Macrophages (CD68, CD163)

    • B cells (CD20)

    • Neutrophils (MPO, CD66b)

  • Analyze spatial relationships between HPRT1-expressing cells and immune infiltrates

  • Quantify co-localization using digital pathology tools

• Correlation analysis with immune signatures:

  • Use CIBERSORT to assess relative abundance of infiltrating immune cell subsets

  • Apply Spearman correlation (recommended filters: cor > 0.3, P < 0.001)

  • Analyze relationship between HPRT1 expression and:

    • StromalScore

    • ImmuneScore

    • ESTIMATEScore

    • TumorPurity

• Functional immunological assays:

  • Co-culture HPRT1-manipulated cancer cells with immune cells

  • Measure immune cell activation, cytokine production, and cytotoxicity

  • Evaluate immune checkpoint molecule expression (PD-L1, CTLA-4)

• Immunomodulator correlation analysis:

  • Examine HPRT1's relationship with three immunomodulator categories:

    • Immunoinhibitors

    • Immunostimulators

    • MHC molecules

  • Generate heatmaps showing correlation patterns

  • Create scatterplots of gene composition with HPRT1

• Integration with TMB and MSI analyses:

  • Assess correlation of HPRT1 with tumor mutational burden (TMB)

  • Evaluate relationship with microsatellite instability (MSI)

  • Use Spearman correlation method for analysis

What techniques can detect post-translational modifications of HPRT1?

Investigating post-translational modifications (PTMs) of HPRT1 requires specialized techniques and antibody applications:

• Phosphorylation-specific analysis:

  • Immunoprecipitation with general HPRT1 antibodies (0.5-4.0 μg per 1-3 mg lysate)

  • Western blot with phospho-specific antibodies

  • Alternative: immunoblotting with general HPRT1 antibodies after:

    • Phosphatase treatment

    • Phos-tag SDS-PAGE for mobility shift detection

  • Mass spectrometry identification of phosphorylation sites

• Ubiquitination detection:

  • Immunoprecipitate HPRT1 using validated antibodies

  • Western blot with anti-ubiquitin antibodies

  • Include proteasome inhibitors in lysis buffer (MG132)

  • Consider denaturing conditions to disrupt protein interactions

• Acetylation analysis:

  • Immunoprecipitate HPRT1

  • Probe with anti-acetylated lysine antibodies

  • Include deacetylase inhibitors in lysis buffer

  • Mass spectrometry validation of acetylation sites

• SUMOylation detection:

  • Lysates prepared with SUMO protease inhibitors (NEM)

  • Immunoprecipitate HPRT1

  • Western blot with anti-SUMO antibodies

  • Validate with mass spectrometry

• Glycosylation analysis:

  • Treat samples with glycosidases (PNGase F, O-glycosidase)

  • Analyze mobility shifts on Western blots

  • Lectin blotting after HPRT1 immunoprecipitation

• Mass spectrometry-based approaches:

  • Immunoprecipitate HPRT1 using highly specific antibodies

  • Trypsin digest and LC-MS/MS analysis

  • Targeted multiple reaction monitoring (MRM) for specific modifications

  • Compare modification patterns between:

    • Normal vs. cancer cells

    • Drug-sensitive vs. resistant cells

While specific PTMs of HPRT1 have not been extensively characterized in the provided research materials, these approaches provide a methodological framework for future investigations that may reveal how modifications regulate HPRT1's role in cancer progression and chemoresistance .

How can HPRT1 antibodies be used for biomarker development?

HPRT1's emerging role in cancer progression positions it as a potential biomarker, with antibodies serving as key tools in biomarker development:

Research has demonstrated that high HPRT1 expression correlates with poor prognosis in nasopharyngeal carcinoma and other cancers , supporting its potential utility as a prognostic biomarker. The standardized use of validated antibodies is essential for consistent biomarker assessment across laboratories and clinical settings.

What are the optimal methods for investigating HPRT1 in nasopharyngeal carcinoma?

Research on HPRT1 in nasopharyngeal carcinoma (NPC) requires specialized methodological approaches:

• Expression analysis in clinical samples:

  • IHC on NPC tissue microarrays using standardized protocols:

    • Recommended antibody dilutions: 1:20-1:16000

    • Antigen retrieval with TE buffer pH 9.0

    • Scoring based on intensity (0-3) and percentage of positive cells (0-3)

  • RT-qPCR and Western blot analysis:

    • Compare HPRT1 levels in NPC vs. nasopharyngeal epithelial cells (e.g., NP460)

    • Validate differential expression in multiple cell lines (e.g., 6-10B, HONE-1)

• Functional studies using knockdown approaches:

  • siRNA-mediated silencing:

    • HPRT1-siRNA3 shows highest knockdown efficiency in NPC cells

    • Verify knockdown by RT-qPCR and Western blot

  • Functional assays after knockdown:

    • Cell viability (MTT assay)

    • Colony formation

    • Wound healing migration assay

    • Transwell invasion assay

• Molecular mechanism investigations:

  • Western blot analysis of downstream targets:

    • Proliferation markers: CyclinD1, CyclinE

    • Chemoresistance markers: MDR1

    • Metastasis markers: MMP-2, MMP-9

  • Signaling pathway analysis:

    • PI3K/AKT pathway components

    • Potential miRNA regulators (miRNA-125b-5p)

• Prognostic analysis:

  • Correlate HPRT1 expression with patient outcomes using Kaplan-Meier survival analysis

  • Multivariate analysis to assess independent prognostic value

  • TCGA data mining for broader validation

Research has demonstrated that HPRT1 is significantly upregulated in NPC compared to normal nasopharyngeal epithelium, and its silencing reduces proliferation, migration, and invasion capabilities of NPC cells . These findings establish HPRT1 as a promising target for further investigation in NPC pathogenesis and potential therapeutic development.

What techniques are most effective for studying HPRT1's role in Lesch-Nyhan syndrome?

Investigating HPRT1 in Lesch-Nyhan syndrome (LNS) requires specialized approaches focusing on loss-of-function scenarios:

• Mutation analysis and functional consequences:

  • Immunohistochemistry to assess HPRT1 protein expression:

    • Use antibody dilutions of 1:20-1:200 for polyclonal or 1:4000-1:16000 for monoclonal antibodies

    • Compare expression in patient vs. control samples

  • Western blot analysis:

    • Detect HPRT1 protein levels and potential truncated forms

    • Use 1:1000-1:10000 dilutions for optimal detection

• Enzymatic activity assays correlated with protein expression:

  • Measure HPRT enzyme activity in patient samples

  • Correlate activity with protein levels detected by antibodies

  • Western blot using HPRT1 antibodies to detect:

    • Wild-type protein (24-28 kDa)

    • Potential mutant forms with altered mobility

• Cellular models of LNS:

  • Patient-derived fibroblasts or lymphoblasts:

    • Immunofluorescence with 1:200-1:1600 dilution

    • Assess localization and expression levels

  • CRISPR/Cas9-engineered cell lines:

    • Validate HPRT1 knockout using antibodies

    • Test rescue with wild-type vs. mutant HPRT1

• Neural implications of HPRT1 deficiency:

  • iPSC-derived neural cultures:

    • Immunofluorescence to assess HPRT1 in different neural cell types

    • Counterstain with neural markers (TUJ1, GFAP, etc.)

  • Brain organoid models:

    • Section and stain for HPRT1 expression patterns

    • Correlate with developmental and functional markers

• Purine metabolism analysis coupled with protein detection:

  • Measure purine metabolites in patient samples

  • Correlate with HPRT1 protein expression quantified by Western blot

  • Analyze salvage pathway components in relation to HPRT1 levels

While the provided search results focus more on HPRT1's role in cancer, these methodological approaches leverage HPRT1 antibodies to investigate the fundamental mechanisms underlying Lesch-Nyhan syndrome, where HPRT1 deficiency leads to severe neurological and behavioral abnormalities .

How can I investigate HPRT1's role in cancer immunotherapy response?

Exploring HPRT1's emerging connection to cancer immunotherapy response requires integrative approaches:

• Correlation of HPRT1 expression with immunotherapy biomarkers:

  • IHC analysis of patient samples:

    • HPRT1 expression (1:20-1:16000 dilution)

    • PD-L1 expression

    • Tumor mutational burden (TMB)

    • Microsatellite instability (MSI)

  • Develop multiplexed staining protocols to visualize:

    • HPRT1-expressing cells

    • Immune cell infiltrates

    • Checkpoint molecule expression

• Immune microenvironment characterization:

  • Apply CIBERSORT to assess immune cell infiltration in relation to HPRT1 expression

  • Correlate HPRT1 with StromalScore, ImmuneScore, and ESTIMATEScore

  • Analyze spatial relationships between HPRT1+ cells and immune populations

  • Quantitative analysis using digital pathology tools

• Functional studies in immunocompetent models:

  • Knockdown or overexpress HPRT1 in cancer cells

  • Implant into immunocompetent mice

  • Treat with immune checkpoint inhibitors

  • Monitor:

    • Tumor growth

    • Immune infiltration

    • Response to immunotherapy

• Cell-based immune response assays:

  • Co-culture HPRT1-modified cancer cells with:

    • T cells

    • NK cells

    • Dendritic cells

  • Measure immune cell activation and cancer cell killing

  • Assess effect of HPRT1 on antigen presentation

• Immunomodulator relationship analysis:

  • Examine correlation between HPRT1 and:

    • Immunoinhibitors

    • Immunostimulators

    • MHC molecules

  • Validate correlations with protein-level analysis using antibodies

  • Investigate functional consequences of these relationships

Research has shown that HPRT1 expression correlates with immune cell infiltration patterns in various cancers, suggesting potential involvement in immunotherapy response mechanisms . These methodological approaches provide a framework for investigating how HPRT1 might influence response to immune checkpoint inhibitors and other immunotherapeutic strategies.

What protocols are recommended for investigating HPRT1 in breast cancer?

For comprehensive investigation of HPRT1 in breast cancer:

Research indicates that HPRT1 RNA levels are significantly elevated in breast cancer tissues, particularly in basal cells and triple-negative breast cancer, suggesting its involvement in cancer progression through positive regulation of genes associated with cancer pathways . These protocols provide a structured approach to further elucidate HPRT1's role in breast cancer biology and potential therapeutic targeting.

What are the key considerations when using HPRT1 antibodies in neurodegenerative disease research?

When applying HPRT1 antibodies to neurodegenerative disease research:

• Optimal tissue preparation for neural tissues:

  • Post-mortem tissue considerations:

    • Account for post-mortem interval effects on HPRT1 epitopes

    • Optimize fixation (4% paraformaldehyde, 24-48 hours)

    • Test multiple antigen retrieval methods:

      • TE buffer pH 9.0 (recommended)

      • Citrate buffer pH 6.0

  • Perfusion-fixed animal tissues:

    • Transcardial perfusion with 4% paraformaldehyde

    • Post-fixation (4-24 hours depending on tissue size)

    • Cryoprotection and sectioning protocols

• Cell type-specific analysis in neural tissues:

  • Double immunofluorescence with:

    • HPRT1 antibodies (1:200-1:1600)

    • Neural cell type markers:

      • Neurons (NeuN, MAP2)

      • Astrocytes (GFAP)

      • Microglia (Iba1)

      • Oligodendrocytes (Olig2)

  • Regional analysis of HPRT1 expression:

    • Cortex

    • Hippocampus

    • Striatum (particularly relevant for Lesch-Nyhan syndrome)

    • Substantia nigra

• Purine metabolism context in neurodegeneration:

  • Connect HPRT1 expression to purine metabolism:

    • Double labeling with purine metabolism enzymes

    • Correlation with purine metabolite levels

  • Investigate HPRT1 in oxidative stress responses:

    • Co-labeling with oxidative stress markers

    • Relationship to antioxidant systems

• Technical adaptations for neural tissue:

  • Higher antibody concentrations may be needed (1:20-1:200)

  • Extended incubation times (overnight at 4°C)

  • Background reduction strategies:

    • Mouse-on-mouse blocking for mouse antibodies on mouse tissue

    • Autofluorescence quenching (Sudan Black B)

    • Lipofuscin autofluorescence reduction

• Models for studying HPRT1 in neurodegeneration:

  • Primary neuron cultures:

    • Optimize fixation (4% PFA, 15 minutes)

    • Use lower antibody dilutions (start with 1:200)

  • iPSC-derived neurons:

    • Patient-derived lines with HPRT1 mutations

    • CRISPR-engineered lines with specific mutations

  • Animal models:

    • HPRT1 knockout/knockdown

    • Neurodegenerative disease models

While HPRT1 is traditionally studied in Lesch-Nyhan syndrome , these methodological approaches facilitate investigation of its potential roles in other neurodegenerative conditions, leveraging the specificity and versatility of available HPRT1 antibodies.