HPRT PAT1D9AT Antibody

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

HPRT (Hypoxanthine-guanine phosphoribosyltransferase) is a critical enzyme in the purine salvage pathway that plays a central role in the generation of purine nucleotides through the conversion of guanine to guanosine monophosphate and hypoxanthine to inosine monophosphate. It functions by transferring the 5-phosphoribosyl group from 5-phosphoribosylpyrophosphate onto the purine . This process is essential for DNA repair and cellular proliferation, making HPRT a fundamental component of cellular metabolism. Mutations in the HPRT1 gene cause Lesch-Nyhan syndrome, a severe neurological disorder, and HPRT deficiency is associated with gout development.

In cancer research, HPRT has gained significant attention as high HPRT1 expression correlates with poor survival in multiple cancer types, including lung adenocarcinoma, head-neck squamous cell carcinoma, and oral squamous cell carcinoma. Furthermore, HPRT1 has been implicated in chemoresistance mechanisms, particularly promoting cisplatin resistance in oral cancer by activating the PI3K/AKT pathway. Recent studies have also established connections between HPRT1 expression and immune checkpoint regulation, with positive correlations observed between HPRT1 and PD-1 levels in head and neck cancers.

As a housekeeping gene with critical metabolic functions and emerging roles in disease mechanisms, HPRT represents an important research target across multiple disciplines, from basic molecular biology to translational cancer research.

What are the specific characteristics of the PAT1D9AT HPRT antibody?

The PAT1D9AT HPRT antibody is a mouse monoclonal antibody specifically designed to recognize human HPRT protein. Its detailed characteristics are as follows:

The antibody has been raised against a recombinant protein representing amino acids 1-218 of human HPRT . This comprehensive coverage of the HPRT protein ensures robust recognition of the target. The PAT1D9AT clone is purified using Protein A chromatography from mouse ascitic fluids, resulting in a highly pure antibody preparation . Its IgG2b isotype with kappa light chains provides good stability and consistent performance across multiple experimental applications.

What applications has the PAT1D9AT HPRT antibody been validated for?

The PAT1D9AT HPRT antibody has been validated for multiple research applications, making it a versatile tool for HPRT-focused investigations. The validated applications include:

This antibody demonstrates specific reactivity with human samples across these applications . For Western blotting, the antibody detects the HPRT protein at its expected molecular weight of 24-28 kDa. Its validation for flow cytometry makes it suitable for quantitative assessment of HPRT expression at the single-cell level, while its compatibility with ICC/IF enables visualization of HPRT's subcellular localization.

Based on general recommendations for HPRT antibodies, researchers typically use the following dilution ranges, although optimal concentrations should be determined empirically for each experimental setup:

• Western Blot: 1:500–1:10,000

• Immunofluorescence: 1:200–1:800

• Flow Cytometry (Intracellular): 0.4 μg/10^6 cells

The multi-application validation of PAT1D9AT makes it a versatile tool for researchers investigating HPRT across different experimental contexts.

How should the PAT1D9AT HPRT antibody be stored and handled for optimal performance?

Proper storage and handling of the PAT1D9AT HPRT antibody are essential for maintaining its activity and specificity over time. The recommended storage and handling conditions are:

For optimal antibody performance and longevity, several practical steps should be implemented upon receiving the antibody . First, it is highly recommended to aliquot the stock solution into smaller volumes to minimize repeated freeze/thaw cycles, which can degrade antibody performance over time. Working dilutions should be prepared fresh and can be stored at 4°C if they will be used within one week.

The antibody formulation contains 10% glycerol, which helps maintain stability during freeze/thaw cycles, and 0.02% sodium azide as a preservative . Researchers should note that sodium azide is toxic and should be handled with appropriate precautions. Additionally, sodium azide can inhibit horseradish peroxidase (HRP) activity, so dilute working solutions sufficiently when using HRP-based detection systems.

Proper storage and handling practices are crucial for ensuring consistent antibody performance across experiments and extending the useful life of this valuable research reagent.

What are the recommended controls when using PAT1D9AT HPRT antibody for quantitative analyses?

Implementing appropriate controls is essential for generating reliable and reproducible data when using the PAT1D9AT HPRT antibody. The following comprehensive control strategy is recommended:

Antibody Specificity Controls:

• Isotype Controls: Use mouse IgG2b isotype controls, specifically Mouse IgG2b [PLRV219] (A86740) or Mouse IgG2b [MPC-11] (A86389), at the same concentration as the PAT1D9AT antibody .

• Knockdown/Knockout Controls: When available, include samples with HPRT knockdown or knockout to confirm antibody specificity.

• Peptide Competition: Pre-incubate the antibody with excess immunizing peptide (recombinant HPRT) to block specific binding sites.

Application-Specific Technical Controls:

For Western Blotting:

• Loading Controls: Include housekeeping proteins (β-actin, GAPDH) or total protein staining methods.

• Positive Control Lysates: Use HeLa, A-431, MCF-7, or HEK-293 cell lysates as positive controls for HPRT expression .

• Molecular Weight Markers: Include on every blot to confirm the expected 24-28 kDa size of HPRT.

For Flow Cytometry:

• Unstained Controls: Essential for setting voltage and determining autofluorescence levels.

• Secondary-Only Controls: When using unconjugated PAT1D9AT, include samples with secondary antibody alone.

• Single-Stained Controls: Critical for compensation when performing multicolor flow cytometry.

• Fixation/Permeabilization Controls: Compare staining in fixed/permeabilized cells versus unfixed cells.

For Immunocytochemistry/Immunofluorescence:

• No-Primary Controls: Samples processed with secondary antibody only to determine background fluorescence.

• Subcellular Marker Co-staining: Include markers for relevant subcellular compartments.

• Counterstains: Use nuclear counterstains (DAPI, Hoechst) to facilitate cellular identification.

Quantification and Normalization Controls:

• Standard Curves: When available, include recombinant HPRT protein standards for absolute quantification.

• Inter-assay Calibrators: Include common calibrator samples across multiple experiments.

• Dynamic Range Controls: Include samples with known high, medium, and low HPRT expression.

Biological Reference Controls:

• Normal Tissue/Cell Controls: Include non-cancerous counterparts when studying cancer cells.

• Treatment Reference Controls: Include untreated samples when studying drug effects.

• Time Point References: For time-course studies, include appropriate time-matched controls.

Implementing this comprehensive control strategy enhances the reliability, specificity, and reproducibility of quantitative data generated using the PAT1D9AT HPRT antibody across various applications.

How can I optimize the PAT1D9AT HPRT antibody for flow cytometry applications?

Optimizing the PAT1D9AT HPRT antibody for flow cytometry requires careful consideration of several methodological aspects, particularly since HPRT is an intracellular protein requiring effective permeabilization strategies:

Sample Preparation and Fixation/Permeabilization:
Since HPRT is an intracellular protein, effective permeabilization is crucial for antibody access. For adherent cells, begin by detaching cells (typically using trypsin), washing in PBS, and then fixing with 4% paraformaldehyde for 10-15 minutes at room temperature. Follow fixation with permeabilization using 0.1-0.5% Triton X-100 or commercially available permeabilization buffers. For suspension cells, collect cells, wash in PBS, and proceed with the same fixation/permeabilization protocol. Methanol fixation can be an alternative approach that combines fixation and permeabilization in one step, which may be advantageous for certain applications.

Controls for Varying Expression Levels:
Include appropriate positive controls, such as cell lines known to express high levels of HPRT. Most human cell lines express HPRT, with HeLa cells commonly used as a positive control . For negative controls, use isotype controls matching the PAT1D9AT antibody's mouse IgG2b isotype. Suitable isotype controls include Mouse IgG2b [PLRV219] (A86740) and Mouse IgG2b [MPC-11] (A86389) .

Secondary Antibody Selection:
If using the unconjugated PAT1D9AT primary antibody, select an appropriate fluorochrome-conjugated secondary antibody. Compatible secondary antibodies include Goat Anti-Mouse IgG H&L Antibody (FITC) (A301669) or other anti-mouse IgG secondaries appropriate for flow cytometry . Match the brightness of the fluorochrome to the expected expression level of HPRT in your samples.

Data Analysis Considerations:
For comparative studies across cell lines with varying expression levels, consider using median fluorescence intensity (MFI) ratios rather than raw MFI values to normalize data. Include unstained and single-stained controls for compensation when performing multicolor flow cytometry. When analyzing changes in HPRT expression under different experimental conditions, consistent gating strategies are essential for reliable comparison.

By systematically optimizing these parameters, researchers can establish robust flow cytometry protocols using the PAT1D9AT HPRT antibody, enabling quantitative assessment of HPRT expression at the single-cell level.

How can the PAT1D9AT HPRT antibody be used in multiplex immunofluorescence studies?

Incorporating the PAT1D9AT HPRT antibody into multiplex immunofluorescence studies requires careful planning to ensure compatibility with other antibodies and optimal signal detection. The following methodological considerations are essential for successful multiplex experiments:

Antibody Compatibility Planning:
Since PAT1D9AT is a mouse-derived antibody (IgG2b isotype with kappa light chains), it's crucial to avoid using other primary antibodies from mouse with the same isotype to prevent cross-reactivity . When designing multiplex panels, prioritize combining antibodies from different host species (rabbit, goat, etc.) with PAT1D9AT. If multiple mouse antibodies must be used, consider directly conjugated versions or implement sequential detection methods with thorough blocking between steps.

Secondary Antibody Selection:
Select secondary antibodies with minimal spectral overlap when designing multiplex panels. For PAT1D9AT, compatible secondary antibodies include Goat Anti-Mouse IgG H&L Antibody (FITC) (A301669) or alternatives with different fluorophores to fit within your multiplex panel design . Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity, which is particularly important in multiplex settings where multiple secondary antibodies are present simultaneously.

Staining Approach Selection:
For multiplex studies involving PAT1D9AT, two main approaches can be considered. Simultaneous staining involves applying all primary antibodies together, followed by all secondary antibodies, which is faster but requires antibodies from different host species to avoid cross-reactivity. Sequential staining completes staining with first primary-secondary pair, followed by blocking, then proceeding with the next pair. This approach reduces cross-reactivity issues and is recommended when using multiple antibodies from the same species or isotype, though it requires more time and sample manipulation.

Protocol Optimization for HPRT Detection:
HPRT is an intracellular enzyme requiring effective permeabilization for antibody access. Test different permeabilization methods (Triton X-100, Saponin, methanol) to determine compatibility with other targets in your multiplex panel. Include appropriate blocking steps (5-10% normal serum from secondary antibody host species) to minimize background fluorescence, which becomes increasingly important as more antibodies are included in the panel.

Technical Controls for Multiplex Immunofluorescence:
Include comprehensive controls for multiplex studies: single-color controls for spectral unmixing if using confocal microscopy; no-primary antibody controls to assess secondary antibody background; isotype controls (Mouse IgG2b [PLRV219] or Mouse IgG2b [MPC-11]) for PAT1D9AT ; positive control samples known to express HPRT (e.g., HeLa cells) ; and co-localization controls if HPRT is expected to co-localize with other proteins of interest.

By carefully addressing these considerations, researchers can successfully incorporate the PAT1D9AT HPRT antibody into multiplex immunofluorescence studies, enabling simultaneous analysis of HPRT expression alongside other proteins of interest.

What approaches can be used to validate PAT1D9AT HPRT antibody specificity in knockout models?

Validating antibody specificity is crucial for generating reliable data, particularly for quantitative studies. For the PAT1D9AT HPRT antibody, several methodological approaches can be implemented to confirm specificity:

Western Blot Validation in Knockout Models:
The gold standard for antibody validation is comparing antibody reactivity between wildtype samples and those with the target protein knocked out. For PAT1D9AT, compare wildtype cells/tissues with HPRT knockout samples using Western blotting. A specific HPRT antibody should show a band at 24-28 kDa in wildtype samples and no band in knockout samples . Include positive control lysates such as HeLa cell lysate, which is known to express HPRT . For optimal resolution, use gradient gels (e.g., 4-20%) to ensure proper separation and visualization of the target protein.

siRNA/shRNA Knockdown Validation:
When knockout models aren't readily available, RNA interference provides an alternative approach for validation. Transfect cells with HPRT-targeting siRNA or shRNA and compare Western blot signal intensity between knockdown and control samples. Quantify band intensity reduction using densitometry and correlate this with qRT-PCR data showing HPRT mRNA reduction. A specific antibody will show proportionate signal reduction corresponding to the knockdown efficiency at both protein and mRNA levels.

Peptide Competition Assays:
Pre-incubate the PAT1D9AT antibody with excess purified HPRT protein or the immunizing peptide before applying to your samples. Run parallel Western blots or immunocytochemistry experiments with both blocked and unblocked antibody. The signal should be significantly reduced or eliminated in samples using the peptide-blocked antibody, confirming that the antibody is binding to its intended epitope rather than interacting non-specifically with other proteins.

Specificity Testing in Multiple Sample Types:
Test the PAT1D9AT antibody across multiple cell lines with known HPRT expression levels. The pattern of detection should align with known expression patterns of HPRT. Based on available information, the HPRT antibody has been validated in multiple human cell lines including HeLa, A-431, MCF-7, and HEK-293 . Consistent detection patterns across diverse sample types provide additional confidence in antibody specificity.

Multi-antibody Approach:
Use multiple HPRT antibodies targeting different epitopes and compare staining patterns and intensity. Consistent results across different antibodies increase confidence in specificity. From the available information, other HPRT antibody clones that could be used for comparison include PAT2G8AT, ARC1300, and various polyclonal antibodies .

These validation approaches provide complementary evidence for antibody specificity, with knockout model validation representing the most definitive proof. By implementing multiple validation strategies, researchers can establish high confidence in the specificity of the PAT1D9AT HPRT antibody, ensuring reliable experimental results.

How does HPRT expression correlate with cancer progression and how can PAT1D9AT be used to study this relationship?

HPRT expression has been linked to cancer progression in multiple tumor types, offering important research opportunities using the PAT1D9AT antibody. According to available data, HPRT1 expression has several significant correlations with cancer progression:

HPRT Expression Patterns in Cancer:
High HPRT1 expression correlates with poor survival in multiple cancers, including lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), and oral squamous cell carcinoma (OSCC). This prognostic relationship makes HPRT an important biomarker for cancer outcomes research. Additionally, HPRT1 has been implicated in chemoresistance mechanisms, particularly promoting cisplatin resistance in OSCC through activation of the PI3K/AKT pathway and upregulation of MMP1. Recent studies have also identified a relationship between HPRT1 and immune regulation, as HPRT1 expression positively correlates with PD-1 levels in HNSC, suggesting potential involvement in immune checkpoint regulation.

Using PAT1D9AT to Investigate HPRT in Cancer:
The PAT1D9AT antibody provides several methodological approaches to investigate these cancer-related phenomena:

Expression Analysis by Western Blotting:
Utilize the PAT1D9AT antibody in Western blot analyses to compare HPRT levels across cancer cell lines with different aggressiveness characteristics. This approach can reveal whether HPRT expression correlates with invasive or metastatic potential. Additionally, Western blotting can be used to correlate HPRT expression with components of the PI3K/AKT pathway, providing mechanistic insights into how HPRT might influence cancer progression. The antibody can also be used to study changes in HPRT expression after drug treatments, potentially identifying therapeutic strategies that modulate HPRT levels.

Flow Cytometry Applications:
The PAT1D9AT antibody's validation for flow cytometry enables quantitative assessment of HPRT expression at the single-cell level . This technique allows researchers to quantify HPRT expression across heterogeneous cancer cell populations and perform dual staining with cell cycle markers to investigate relationships between HPRT expression and proliferation. Flow cytometry also facilitates correlation of HPRT levels with stem cell or differentiation markers, potentially identifying specific subpopulations with altered HPRT expression.

Immunofluorescence Applications:
Using PAT1D9AT in immunofluorescence studies allows researchers to assess the subcellular localization of HPRT in cancer cells, which may change during disease progression . Co-staining with proliferation markers (Ki-67) or other cancer-related proteins can reveal spatial relationships between HPRT and other cancer-associated factors. This approach is particularly valuable for examining expression patterns in heterogeneous tumor cell populations.

Methodological Considerations:
When using PAT1D9AT to study HPRT in cancer, several methodological aspects should be considered. Include appropriate normal tissue/cell counterparts as controls to establish baseline HPRT expression. Consider genetic manipulation approaches (knockdown/overexpression) to validate functional correlations between HPRT and cancer phenotypes. For quantification, use normalized densitometry against housekeeping proteins for Western blot, mean/median fluorescence intensity analysis for flow cytometry, and integrated density measurements for immunofluorescence.

By leveraging the PAT1D9AT antibody across these applications and following these methodological considerations, researchers can effectively investigate the role of HPRT in cancer progression, potentially identifying new therapeutic targets or prognostic markers.

How can the PAT1D9AT HPRT antibody be used to investigate chemoresistance mechanisms?

The PAT1D9AT HPRT antibody provides valuable methodological approaches for investigating the relationship between HPRT expression and chemoresistance, an emerging area of research interest. According to available information, HPRT1 has been implicated in promoting cisplatin resistance in oral squamous cell carcinoma through activation of the PI3K/AKT pathway and upregulation of MMP1. Several experimental strategies utilizing the PAT1D9AT antibody can be implemented to further explore these mechanisms:

Establishing and Characterizing Resistant Cell Models:
Develop chemoresistant cell lines through gradual exposure to increasing drug concentrations, mimicking the clinical development of resistance. Use the PAT1D9AT antibody in Western blot analysis to compare HPRT expression between parental and resistant cell lines, quantifying expression differences using densitometry normalized to loading controls. This approach can establish whether HPRT upregulation is a consistent feature of resistance development across different cancer types or drug treatments.

In parallel, create HPRT overexpression and knockdown models to directly test the functional role of HPRT in drug response. Confirm expression changes using the PAT1D9AT antibody in Western blot and flow cytometry applications, then assess how these genetic modifications alter sensitivity to chemotherapeutic agents through standard viability assays. This genetic approach provides causal evidence for HPRT's role in resistance mechanisms.

Investigating PI3K/AKT Pathway Connections:
To explore the reported connection between HPRT and the PI3K/AKT pathway, use PAT1D9AT for HPRT expression quantification alongside antibodies targeting phosphorylated forms of AKT, mTOR, and other pathway components. Correlate HPRT levels with pathway activation markers across sensitive and resistant cell models. Additionally, monitor changes in both HPRT expression and pathway activation during resistance development to establish temporal relationships between these events.

Complement correlative studies with functional investigations by assessing how PI3K/AKT inhibitors affect HPRT expression. Determine if HPRT knockdown sensitizes cells to PI3K/AKT inhibition, potentially identifying synergistic therapeutic approaches. Use the PAT1D9AT antibody in Western blot and flow cytometry to monitor HPRT levels throughout these intervention studies.

Flow Cytometry-Based Resistance Studies:
Leverage the PAT1D9AT antibody's validation for flow cytometry to perform multi-parameter analyses of drug response at the single-cell level. Combine PAT1D9AT antibody with markers of apoptosis (Annexin V/PI) after drug treatment to determine if cells with higher HPRT expression show differential survival following chemotherapy exposure. This approach can reveal whether HPRT expression levels directly correlate with cell survival under drug pressure.

The flow cytometry application also enables sorting strategies where the PAT1D9AT antibody is used to isolate cells based on HPRT expression levels. These sorted populations can then be tested for drug sensitivity and analyzed for differences in PI3K/AKT pathway activation, providing functional evidence for HPRT's role in resistance mechanisms.

Drug Sensitivity Correlation Studies:
Available information indicates that HPRT1 has been linked to drug sensitivity profiles, with identification of 16 drugs targeting HPRT1 in lung and oral cancers. To expand on this finding, screen a panel of cancer cell lines for HPRT expression using the PAT1D9AT antibody and correlate expression levels with IC50 values for various chemotherapeutic agents. This approach can identify both positive and negative correlations between HPRT levels and drug sensitivity, potentially revealing novel drug vulnerabilities in HPRT-high or HPRT-low cancers.

By implementing these methodological approaches with the PAT1D9AT HPRT antibody, researchers can comprehensively investigate the role of HPRT in chemoresistance mechanisms, potentially identifying new therapeutic strategies for overcoming treatment resistance in cancer patients.

How does the PAT1D9AT HPRT antibody compare to other commercially available HPRT antibodies?

The PAT1D9AT HPRT antibody represents one of several commercially available options for HPRT detection, each with distinct characteristics that may influence selection for specific research applications. Understanding these differences enables researchers to make informed choices based on their experimental requirements:

Clonal Diversity and Host Species:
The PAT1D9AT clone is a mouse monoclonal antibody with IgG2b isotype and kappa light chains . Other available HPRT antibody options include the mouse monoclonal PAT2G8AT clone (IgG1 isotype), the rabbit monoclonal ARC1300 clone, and various rabbit polyclonal antibodies . This diversity in host species and clonality provides flexibility for multiplex studies where antibody compatibility is essential. When designing co-staining experiments, researchers can select HPRT antibodies from different host species to avoid cross-reactivity with secondary detection systems.

Application Versatility:
While PAT1D9AT is validated for Western blot, ELISA, ICC/IF, and flow cytometry with human samples , other HPRT antibodies offer complementary application profiles. For instance, some rabbit polyclonal antibodies are specifically validated for immunohistochemistry (IHC) applications . The comparative application profiles are summarized in the following table:

This application diversity allows researchers to select antibodies optimized for their specific experimental techniques. For example, if tissue immunohistochemistry is required, a rabbit polyclonal antibody validated for IHC might be preferable to PAT1D9AT.

Species Reactivity:
PAT1D9AT is specifically reactive with human samples , while some other antibodies offer broader species reactivity, including mouse and rat samples . For comparative studies across species, researchers might select antibodies like the rabbit monoclonal ARC1300 clone, which reacts with human, mouse, and rat samples . This consideration is particularly important for researchers working with animal models where cross-species reactivity is essential.

Validation Status:
Different HPRT antibodies have varying levels of validation. Some have been knockout (KO) validated to confirm their specificity to HPRT , representing the gold standard in antibody validation. When selecting an antibody for critical experiments requiring high confidence in specificity, researchers should prioritize clones with extensive validation documentation.

Pricing and Sizing Options:
The PAT1D9AT antibody is available in various size options (5μg, 20μg, 100μg) , providing flexibility for different experimental scales. Other HPRT antibodies may be available in different volume or quantity formats (e.g., 100μl) . This sizing diversity allows researchers to select appropriate amounts based on their experimental needs and budget constraints.

By considering these comparative aspects, researchers can select the most appropriate HPRT antibody for their specific experimental requirements, whether prioritizing application versatility, species reactivity, validation status, or other factors critical to their research objectives.

What methodological approaches are recommended for quantitative Western blot analysis using PAT1D9AT?

Quantitative Western blot analysis using the PAT1D9AT HPRT antibody requires careful methodological considerations to ensure accurate and reproducible results. The following comprehensive approach addresses key aspects of experimental design, technical implementation, and data analysis:

Sample Preparation Optimization:
Begin with standardized protein extraction using buffers compatible with HPRT preservation. Since HPRT is a cytosolic protein, standard RIPA or NP-40 based lysis buffers are typically suitable. Determine optimal protein loading through preliminary experiments, typically starting with 20-30 μg of total protein for cell lysates based on successful detection in HeLa, A-431, MCF-7, and HEK-293 cell lysates . Include protease inhibitor cocktails in lysis buffers to prevent protein degradation, and standardize protein quantification methods (BCA or Bradford) across all samples.

Gel Electrophoresis and Transfer Parameters:
Select appropriate percentage gels based on HPRT's molecular weight of 24-28 kDa—typically 12-15% polyacrylamide gels provide optimal resolution for proteins in this size range. For transfer optimization, consider using PVDF membranes with 0.2 μm pore size rather than 0.45 μm to better retain smaller proteins like HPRT. Implement wet transfer systems with methanol-containing buffers, typically at 100V for 1 hour or 30V overnight at 4°C, to ensure efficient transfer of the target protein.

Antibody Incubation and Detection Optimization:
Determine the optimal PAT1D9AT antibody dilution through systematic titration experiments, starting with the manufacturer's recommendations and adjusting based on signal intensity and background levels. Typically, include a range of dilutions (e.g., 1:500, 1:1000, 1:2000) to identify the optimal concentration for your specific experimental system. For signal detection, select an appropriate secondary antibody, such as Goat Anti-Mouse IgG H&L Antibody (HRP) (A301445) , and optimize its dilution in parallel.

Controls for Quantitative Analysis:
Implement a comprehensive control strategy for quantitative Western blotting. Include technical replicates (minimum triplicate) to assess consistency and biological replicates across independent experiments to establish biological significance. For normalization, include appropriate loading controls such as β-actin or GAPDH, ensuring that these proteins are not affected by your experimental conditions. Additionally, incorporate standard curves using recombinant HPRT protein (when available) to establish the linear range of detection and facilitate absolute quantification.

Quantification and Statistical Analysis:
For reliable quantification, capture images using a digital imaging system with a broad dynamic range, ensuring signal is not saturated. Use software that enables background subtraction and normalization to loading controls (e.g., ImageJ, Image Lab). Export numerical data for statistical analysis in appropriate software (GraphPad Prism, R, SPSS), applying suitable statistical tests based on your experimental design. For comparative studies, consider presenting data as fold change relative to control samples rather than absolute values to account for inter-blot variability.

Troubleshooting Common Issues:
Several common issues may arise during quantitative Western blotting with PAT1D9AT. If signal is weak, consider longer primary antibody incubation (overnight at 4°C), increased antibody concentration, or more sensitive detection systems. For high background, implement more stringent washing steps, increase blocking time, or reduce secondary antibody concentration. If multiple bands appear, optimize transfer conditions and consider longer blocking times or alternative blocking agents. For inconsistent loading control signals, ensure equal loading volume and consistent transfer efficiency across the gel.

By implementing these methodological considerations, researchers can generate reliable quantitative Western blot data using the PAT1D9AT HPRT antibody, enabling accurate comparison of HPRT expression across experimental conditions.