PRPS2 Antibody

What is PRPS2 and what is its significance in cellular metabolism?

PRPS2 is an enzyme that catalyzes the synthesis of phosphoribosyl pyrophosphate (PRPP), which is essential for nucleotide synthesis . It belongs to a family of phosphoribosyl pyrophosphate synthetase proteins, with three isoforms encoded by different genes in humans . PRPS2 plays a crucial role in cellular metabolism by providing the PRPP substrate necessary for both purine and pyrimidine nucleotide biosynthesis pathways. Research has demonstrated that PRPS2 is particularly important in highly proliferative contexts, where demand for nucleotide synthesis is elevated. The protein has a molecular weight of approximately 30-34 kDa and is detected in various tissues including testis, spleen, and multiple cell lines .

What are the typical applications for PRPS2 antibodies in research?

PRPS2 antibodies have been validated for multiple research applications, each providing unique insights into PRPS2 biology:

These applications have been documented in multiple publications, demonstrating the versatility of PRPS2 antibodies as research tools . The specific dilutions and conditions may need optimization based on the particular experimental system and antibody used.

What species reactivity has been confirmed for commercially available PRPS2 antibodies?

Commercial PRPS2 antibodies have been extensively tested and validated for reactivity with specific species:

Both the tested reactivity and cited reactivity in publications indicate successful detection in these species . This cross-species reactivity makes these antibodies versatile tools for comparative studies between human and murine models. The high sequence homology of PRPS2 between species contributes to this cross-reactivity, though researchers should always validate new sample types before proceeding with full-scale experiments.

What are the optimal sample preparation protocols for PRPS2 detection in Western blot?

For optimal PRPS2 detection by Western blot, the following protocol elements are critical:

• Sample preparation:

  • Use lysis buffers containing protease inhibitors to prevent degradation

  • 10% SDS-PAGE gels provide appropriate separation for the 30-34 kDa PRPS2 protein

  • Complete denaturation is essential; samples should be heated at 95°C for 5 minutes in sample buffer

• Transfer and blocking:

  • Standard transfer protocols are effective for PRPS2

  • Blocking with either 5% non-fat milk or BSA in TBS-T (20-60 minutes at room temperature)

• Antibody incubation:

  • Primary antibody dilution: 1:500-1:2000 in blocking buffer

  • Overnight incubation at 4°C typically yields optimal results

  • HRP-conjugated secondary antibody appropriate to the primary antibody host species

• Positive controls:

  • A375 cells, HepG2 cells, and testicular tissue lysates show consistent PRPS2 expression

The expected molecular weight for PRPS2 is 30-34 kDa, and researchers should look for specific bands in this range when validating their Western blot results .

How should antigen retrieval be optimized for PRPS2 immunohistochemistry?

Antigen retrieval is a critical step for successful PRPS2 detection in immunohistochemistry. The search results indicate specific protocols have been validated:

• Primary recommendation:

  • TE buffer (10 mM Tris, 1 mM EDTA) at pH 9.0

  • Heat-induced epitope retrieval (95-98°C for 15-20 minutes)

  • Allow gradual cooling to room temperature (20 minutes)

• Alternative method:

  • Citrate buffer (10 mM sodium citrate) at pH 6.0

  • Similar heating and cooling parameters as above

The choice between these methods may depend on:

• Tissue type and fixation duration

• Multiplex staining requirements

• Equipment availability

For human stomach tissue, which serves as a positive control, the TE buffer method (pH 9.0) is specifically recommended . Researchers should titrate the PRPS2 antibody concentration (typically 1:1000-1:4000) after optimizing the antigen retrieval method to achieve the best signal-to-background ratio.

What are the best fixation and permeabilization methods for PRPS2 immunofluorescence?

For optimal PRPS2 detection by immunofluorescence, the following fixation and permeabilization protocols have been validated:

• Cell culture fixation options:

  • 95% ethanol fixation for 30 minutes (recommended method)

  • Alternative: 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilization:

  • 0.4% Triton X-100 for 15 minutes

  • This concentration has been specifically validated for PRPS2 detection

• Blocking conditions:

  • 10% fetal bovine serum (FBS) at room temperature for 20 minutes

  • Alternative: 1-5% BSA in PBS if serum cross-reactivity is a concern

• Antibody application:

  • PRPS2 antibody dilution range: 1:200-1:800

  • Incubation at 4°C overnight for optimal results

These conditions have been successfully employed in studies examining PRPS2 localization in cell lines (A375, GC1, GC2) and tissue sections . The protocol yields specific staining with minimal background, allowing clear visualization of PRPS2's subcellular distribution.

How can I verify the specificity of my PRPS2 antibody results?

Verifying PRPS2 antibody specificity requires multiple validation approaches:

• Positive controls:

  • Cell lines: A375, HepG2, GC1, and GC2 cells have confirmed PRPS2 expression

  • Tissues: Mouse spleen, human stomach tissue, and testicular tissue show reliable PRPS2 expression

• Negative controls:

  • PRPS2 knockdown or knockout samples:

    • Published studies have used shRNA-mediated PRPS2 depletion successfully

    • These serve as critical specificity controls

  • Primary antibody omission: Replace primary antibody with the same concentration of non-immune IgG from the same species

• Multiple detection methods:

  • Confirm key findings with more than one application (e.g., WB and IHC)

  • When possible, use antibodies targeting different PRPS2 epitopes

• Expected results:

  • Western blot: Single band at 30-34 kDa

  • IHC/IF: Pattern consistent with cytoplasmic expression

  • Correlation with mRNA expression data where available

Implementing these validation steps increases confidence in the specificity of observed PRPS2 signals and helps distinguish true results from potential artifacts.

What strategies can resolve high background or non-specific binding with PRPS2 antibodies?

When encountering high background or non-specific binding with PRPS2 antibodies, consider these optimization strategies:

• Antibody dilution optimization:

  • Test a dilution series spanning the recommended range

  • For Western blot: 1:500-1:2000

  • For IHC: 1:1000-1:4000

  • For IF/ICC: 1:200-1:800

• Blocking protocol modifications:

  • Increase blocking time (30-60 minutes)

  • Try alternative blocking agents (BSA vs. serum vs. commercial blockers)

  • For challenging samples, include 0.1-0.3% Triton X-100 in blocking buffer

• Washing improvements:

  • Increase wash buffer volume

  • Extend washing times (5-6 washes of 5-10 minutes each)

  • Add 0.1-0.2% Tween-20 to wash buffers

• Sample-specific optimizations:

  • For IHC: Optimize antigen retrieval (pH 9.0 TE buffer is recommended)

  • For IF: Ensure proper permeabilization (0.4% Triton X-100 for 15 minutes)

  • For WB: Consider alternative membrane blocking methods

• Antibody absorption:

  • Pre-absorb antibody with tissues or cell lysates known to have high non-specific binding

These approaches have been successful in published studies using PRPS2 antibodies and can help achieve clean, specific signals with minimal background interference.

How can PRPS2 knockdown experiments be designed to study its biological functions?

Designing effective PRPS2 knockdown experiments requires careful consideration of several factors:

• Knockdown technology selection:

  • Transient approaches:

    • siRNA transfection (effective for 48-72 hours)

    • Multiple target sequences recommended to rule out off-target effects

  • Stable approaches:

    • shRNA lentiviral vectors have been successfully used for PRPS2

    • Provides long-term suppression for extended studies

• Essential controls:

  • Non-targeting siRNA/shRNA sequences

  • PRPS2 overexpression rescue experiments

  • Wild-type/untreated controls

• Knockdown validation methods:

  • mRNA level: qRT-PCR (most sensitive method)

  • Protein level: Western blot with PRPS2 antibody (1:500-1:2000)

  • Functional assays: Enzymatic activity measurements where appropriate

• Phenotype assessment:

  • Cell viability and apoptosis assays:

    • Flow cytometry with Annexin V/PI

    • Caspase activation (caspase 6, caspase 9)

  • Proliferation assays

  • Metabolic profiling (nucleotide levels)

  • Transcriptomic analysis

This approach has been successfully employed in studies showing that PRPS2 depletion increases apoptosis in spermatogenic cells (from 2.8% to 16.1% in GC1 cells and from 4.0% to 18.7% in GC2 cells) .

What is the relationship between PRPS2 and apoptotic signaling pathways?

Research has revealed important connections between PRPS2 and apoptotic signaling pathways:

• PRPS2 depletion induces apoptosis:

  • shRNA-mediated PRPS2 knockdown significantly increases apoptotic rates:

    • In GC1 cells: from 2.8% to 16.1%

    • In GC2 cells: from 4.0% to 18.7%

  • Conversely, PRPS2 overexpression reduces apoptosis:

    • In GC1 cells: reduced to 0.5%

    • In GC2 cells: reduced to 1.9%

• Molecular mechanisms:

  • PRPS2 knockdown upregulates pro-apoptotic caspases:

    • Increased expression of Caspase 6

    • Increased expression of Caspase 9

  • PRPS2 regulates E2F1 transcription factor:

    • PRPS2 and E2F1 co-localize in cells

    • PRPS2 knockdown decreases E2F1 expression

    • E2F1 overexpression can partially rescue the apoptotic phenotype in PRPS2-depleted cells

• Physiological relevance:

  • PRPS2 expression is reduced in mouse models of hypospermatogenesis

  • shPRPS2 lentivirus treatment in vivo leads to:

    • Damage to seminiferous tubules

    • Decreased numbers of spermatogenic cells

• Transcriptional effects:

  • RNA-seq analysis identified differential gene expression associated with:

    • Cell cycle regulation

    • Differentiation

    • Apoptosis

    • Cell adhesion

These findings suggest PRPS2 acts as a critical regulator of cell survival, potentially by maintaining nucleotide pools necessary for cellular functions or through direct interactions with apoptotic machinery.

How can PRPS2 antibodies be utilized in protein-protein interaction studies?

PRPS2 antibodies provide valuable tools for investigating protein-protein interactions through several methodologies:

• Co-immunoprecipitation (Co-IP):

  • Protocol recommendations:

    • Use 0.5-4.0 μg of PRPS2 antibody per 1.0-3.0 mg protein lysate

    • Prepare lysates under non-denaturing conditions

    • Pre-clear lysates with protein A/G beads

    • Perform negative controls with non-specific IgG

  • Successfully used to identify PRPS2 interactions

• Immunofluorescence co-localization:

  • Protocol elements:

    • Fix cells with 95% ethanol for 30 minutes

    • Permeabilize with 0.4% Triton X-100 for 15 minutes

    • Block with 10% FBS for 20 minutes

    • Co-incubate with PRPS2 antibody (1:150-1:200 dilution) and antibody against potential interacting protein

  • This approach has successfully demonstrated co-localization of PRPS2 with E2F1 in spermatogenic cells

• Proximity Ligation Assay (PLA):

  • Combines immunofluorescence principles with rolling circle amplification

  • Provides single-molecule resolution of protein interactions

  • Can detect transient interactions in fixed cells/tissues

• Quantitative validation methods:

  • Luciferase reporter assays to confirm functional interactions

  • Knockout/rescue experiments to establish specificity

  • Domain mapping to identify interaction regions

These methods have revealed that PRPS2 interacts with key regulatory proteins such as E2F1, potentially explaining its role in transcriptional regulation and apoptotic signaling pathways .

What experimental approaches can investigate PRPS2 regulation at the post-translational level?

Investigating PRPS2 post-translational regulation requires several complementary experimental approaches:

• Identification of modifications:

  • Mass spectrometry analysis following immunoprecipitation:

    • Use PRPS2 antibody to isolate the protein (0.5-4.0 μg for 1.0-3.0 mg lysate)

    • Analyze purified samples by LC-MS/MS

    • Identify phosphorylation, ubiquitination, acetylation sites

  • Western blot with modification-specific antibodies:

    • Phospho-specific antibodies

    • Ubiquitin antibodies

    • Run samples on Phos-tag gels to separate phosphorylated species

• Stability and turnover analysis:

  • Cycloheximide chase assays:

    • Treat cells with cycloheximide to block new protein synthesis

    • Collect samples at intervals (0-24h)

    • Detect PRPS2 levels by Western blot (1:500-1:2000 dilution)

  • Proteasome/lysosome inhibitor studies:

    • MG132 (proteasome inhibitor) treatment

    • Monitor PRPS2 levels by Western blot

• Enzyme identification:

  • Co-immunoprecipitation to identify interacting kinases/phosphatases

  • Kinase inhibitor screening

  • In vitro kinase assays with purified components

• Functional analysis:

  • Site-directed mutagenesis of modification sites

  • Expression of phosphomimetic or non-phosphorylatable mutants

  • Analysis of mutant effects on:

    • Enzymatic activity

    • Protein-protein interactions

    • Cellular phenotypes

These approaches can provide insights into how PRPS2 activity and function are regulated post-translationally in different cellular contexts, potentially revealing therapeutic intervention points in disease states.

How does PRPS2 expression differ between normal and pathological tissues?

Research has revealed distinct PRPS2 expression patterns in normal versus pathological states:

• Reproductive system pathology:

  • Hypospermatogenesis:

    • Significantly decreased PRPS2 expression in mouse models

    • Reduction in both protein and mRNA levels

    • PRPS2 depletion causes phenotypes resembling hypospermatogenesis:

      • Reduced spermatogenic cells

      • Damaged seminiferous tubules

      • Increased apoptotic rates

• Developmental expression patterns:

  • Normal development:

    • PRPS2 expression in mouse testis gradually increases from 4 to 10 weeks

    • This coincides with maturation of spermatogenic processes

• Cell-type specific expression:

  • Differential expression across tissues:

    • Detectable in specific cell lines (A375, HepG2, GC1, GC2)

    • Present in human stomach tissue

    • Expressed in testicular tissue

These expression differences suggest PRPS2 may serve as a potential biomarker for conditions like hypospermatogenesis and possibly other disorders related to nucleotide metabolism dysregulation. The specific antibody dilutions for detecting these differences are:

• IHC: 1:1000-1:4000

• Western blot: 1:500-1:2000

• Immunofluorescence: 1:200-1:800

How can PRPS2 antibodies contribute to metabolic pathway studies?

PRPS2 antibodies offer valuable tools for investigating nucleotide metabolism pathways through several approaches:

• Enzyme complex identification:

  • Co-immunoprecipitation with other metabolic enzymes:

    • Use 0.5-4.0 μg PRPS2 antibody per 1.0-3.0 mg lysate

    • Identify interactions with nucleotide synthesis enzymes

    • Western blot analysis of precipitated complexes

  • Immunofluorescence co-localization studies:

    • Track spatial organization of metabolic enzyme complexes

    • Detect potential metabolic microdomains

• Regulatory cascade mapping:

  • Chromatin immunoprecipitation:

    • Identify transcription factors controlling PRPS2 expression

    • E2F1 has been shown to co-localize with PRPS2

  • Phosphorylation state analysis:

    • Examine how PRPS2 activity is regulated by phosphorylation

    • Connect to upstream signaling pathways

• Metabolic perturbation studies:

  • PRPS2 knockdown impact on metabolites:

    • Measure nucleotide pools after PRPS2 depletion

    • Trace experiments with labeled precursors

  • Rescue experiments:

    • Re-expression of wildtype vs. mutant PRPS2

    • Monitor restoration of metabolic profiles

• Disease model applications:

  • Tissue expression profiling:

    • IHC analysis (1:1000-1:4000) in disease vs. normal tissues

    • Correlation with metabolic alterations

These approaches can illuminate how PRPS2 functions within nucleotide metabolism networks and how its dysregulation may contribute to pathological states, providing potential targets for therapeutic intervention.

What are the optimal strategies for multiplexing PRPS2 antibodies with other biomarkers?

For successful multiplexing of PRPS2 with other biomarkers, researchers should consider these optimized strategies:

• Antibody panel design:

  • Host species selection:

    • Choose primary antibodies from different host species when possible

    • For example, rabbit anti-PRPS2 with mouse anti-E2F1

  • Isotype consideration:

    • Select different isotypes for same-species antibodies

    • Enables detection with isotype-specific secondary antibodies

• Fluorophore selection for immunofluorescence:

  • Spectral separation:

    • Choose fluorophores with minimal spectral overlap

    • Account for tissue autofluorescence spectrum

  • Signal strength balancing:

    • Match fluorophore brightness to target abundance

    • PRPS2 antibody dilution range: 1:200-1:800

• Chromogenic multiplexing for IHC:

  • Sequential detection methods:

    • Multiple rounds of antibody staining with stripping

    • Tyramide signal amplification for weak signals

  • PRPS2 antibody concentration: 1:1000-1:4000

  • Complementary chromogens with distinct colors

• Validated protocols:

  • Double immunofluorescent staining has successfully demonstrated PRPS2 and E2F1 co-localization:

    • Fix cells with 95% ethanol for 30 minutes

    • Permeabilize with 0.4% Triton X-100 for 15 minutes

    • Block with 10% FBS for 20 minutes

    • Co-incubate with PRPS2 (1:150) and E2F1 (1:200) antibodies overnight at 4°C

• Controls for multiplexing:

  • Single-stain controls to assess bleed-through

  • Secondary-only controls for background determination

  • Absorption controls to verify antibody specificity

These approaches enable researchers to simultaneously visualize PRPS2 with interacting proteins or pathway components, providing insights into functional relationships in complex biological systems.