PRPS1 Antibody

What is PRPS1 and why is it important in cellular metabolism?

PRPS1 catalyzes the phosphoribosylation of D-ribose 5-phosphate (R-5P) to 5-phosphoribosyl-1-pyrophosphate, a crucial step in nucleotide synthesis. This enzyme not only controls purine biosynthesis and supplies precursors for DNA and RNA biosynthesis but also regulates the pentose phosphate pathway (PPP) through a feedback loop involving its substrate R-5P. The enzymatic activity of PRPS1 is essential for cell-cycle regulation, particularly during S phase when nucleotide demand increases for DNA replication . Due to its central role in cellular metabolism, PRPS1 dysfunction has been implicated in several pathological conditions, including cancer where increased activity has been observed.

What species reactivity can be expected with PRPS1 antibodies?

Based on sequence homology analysis, PRPS1 antibodies targeting conserved regions (particularly the N-terminal region) typically show cross-reactivity across multiple species. Commercially available antibodies have predicted reactivity with bovine, human, mouse, primate, rat, and Xenopus samples . This cross-reactivity stems from the high conservation of PRPS1 structure across vertebrate species. When selecting an antibody for your research, it's advisable to verify the specific epitope sequence alignment across your target species to confirm potential reactivity.

What are the common applications for PRPS1 antibodies?

PRPS1 antibodies are primarily used in:

• Western blotting (recommended dilution ~1:1000)

• Immunoprecipitation for protein interaction studies

• Immunohistochemistry for tissue localization

• Activity assays when coupled with enzymatic function tests

For optimized results in Western blotting applications, use purified polyclonal antibodies supplied in PBS with 0.09% (W/V) sodium azide. These antibodies are typically prepared by Saturated Ammonium Sulfate (SAS) precipitation followed by dialysis against PBS .

How should PRPS1 antibodies be stored for maximum stability?

For optimal preservation of antibody function:

• Maintain refrigerated at 2-8°C for short-term storage (up to 2 weeks)

• For long-term storage, keep at -20°C in small aliquots to prevent freeze-thaw cycles

• If lyophilized, reconstitute only the amount needed for immediate use

• Avoid repeated freeze-thaw cycles which can lead to protein denaturation and loss of binding efficiency

How can PRPS1 enzymatic activity be accurately measured in research samples?

PRPS1 activity assessment requires a multi-step procedure involving protein purification and activity detection. The established protocol is as follows:

• Purify PRPS1 from cell lysates (either using antibody-based immunoprecipitation or FLAG-tagged recombinant expression)

• Quantify purified protein via Bradford method

• Incubate purified PRPS1 in reaction buffer containing:

  • 50 mmol/L Tris-HCl, pH 7.4

  • 5 mmol/L MgCl₂

  • 1 mmol/L EDTA

  • 1 mmol/L dithiothreitol

  • 32 mmol/L Na₃PO₄

  • 0.5 mmol/L ATP

  • 0.15 mmol/L ribose 5'-phosphate

  • 0.25 mmol/L P¹P⁵-diadenosine pentaphosphate (Ap5A)

• Incubate at 37°C for 15 minutes

• Terminate reaction with equal volume of 0.1 mol/L EDTA

• Measure AMP concentration via HPLC-based end-point assay

• Calculate AMP/PRPS1 ratio to determine enzymatic activity

For tissue samples, additional homogenization and debris removal via centrifugation is required before antibody purification.

What approaches can detect phosphorylation-dependent regulation of PRPS1?

PRPS1 activity is regulated through phosphorylation, particularly at serine 103 (S103) by CDK1 during the cell cycle. To investigate this regulatory mechanism:

• Immunoprecipitation coupled with mass spectrometry:

  • Purify PRPS1 (tagged or using specific antibodies) from cells at different cell cycle stages

  • Perform tryptic digestion of gel-separated PRPS1

  • Analyze peptides by LC/MS-MS (e.g., on an Orbitrap-XL mass spectrometer)

  • Identify phosphopeptides using database search tools like Mascot or Sequest

  • Confirm phosphorylation sites using PhosphoRS or similar phosphosite localization software

• In vitro kinase assays:

  • Incubate purified recombinant PRPS1 (e.g., GST-PRPS1) with purified kinase (e.g., His-CDK1)

  • Use kinase buffer containing: 50 mmol/L Tris-HCl pH 7.5, 100 mmol/L KCl, 50 mmol/L MgCl₂, 1 mmol/L Na₃VO₄, 1 mmol/L DTT, 5% glycerol, and 0.05 mmol/L ATP

  • Incubate at 25°C for 1 hour

  • Analyze by SDS-PAGE and detect using phospho-specific antibodies

• Phospho-specific antibodies:

  • Use antibodies specifically detecting PRPS1 phosphorylation at S103

  • Apply in Western blotting or immunohistochemistry to assess phosphorylation status

  • Compare results across different experimental conditions or clinical samples

How can PRPS1 antibodies be validated for specificity in experimental systems?

Thorough validation of PRPS1 antibodies is crucial for reliable research outcomes. A comprehensive validation approach includes:

• Western blot analysis with positive and negative controls:

  • Positive controls: HeLa cells (confirmed to express PRPS1 by BioGPS gene expression data)

  • Negative controls: PRPS1 knockout cells or tissues

  • Expected molecular weight: ~34.8 kDa

• Antibody specificity testing:

  • Pre-adsorption with immunizing peptide should abolish signal

  • Cross-reactivity assessment with related isoforms (PRPS2, PRPS1L1)

  • Validation across multiple cell lines with known PRPS1 expression profiles

• Knockdown/knockout verification:

  • siRNA or CRISPR-based PRPS1 depletion should reduce or eliminate antibody signal

  • Complementation with ectopic expression should restore signal

• Epitope mapping:

  • Confirm antibody recognizes the expected region (e.g., N-terminal region between amino acids 79-109 for certain antibodies)

  • Test against truncated proteins to verify binding site

How does PRPS1 activity correlate with colorectal cancer progression?

Research has revealed significant correlations between PRPS1 activity and colorectal cancer:

• PRPS1 enzymatic activity is significantly higher in colorectal cancer tissues compared to adjacent normal tissues, even when mRNA and protein level differences are modest

• Clinical correlation analysis with 184 patients demonstrated:

  • Higher PRPS1 enzymatic activity levels in more advanced colorectal cancer cases

  • Positive correlation between PRPS1 activity and cancer recurrence

• PRPS1 phosphorylation at S103 (detected using phospho-specific antibodies) serves as a promising diagnostic biomarker for colorectal cancer

This suggests that PRPS1 activity, rather than just expression level, may be a more relevant parameter for cancer progression assessment.

What methodological approaches can detect PRPS1 activity differences between normal and cancerous tissues?

To accurately measure and compare PRPS1 activity between normal and cancerous tissues:

• Tissue preparation:

  • Collect matched tumor and adjacent normal tissues

  • Homogenize in appropriate lysis buffer

  • Remove debris by high-speed centrifugation

  • Quantify total protein concentration

• PRPS1 purification:

  • Immunoprecipitate PRPS1 using specific antibodies

  • Alternatively, use tagged PRPS1 expression systems

• Activity assay:

  • Perform enzyme activity assay as described in question 2.1

  • Calculate AMP/PRPS1 ratio to determine specific activity

  • Compare values between normal and tumor tissues

• Phosphorylation analysis:

  • Detect S103 phosphorylation status using phospho-specific antibodies

  • Compare phosphorylation levels between normal and tumor samples

What are the critical controls needed when studying PRPS1 phosphorylation in cell cycle regulation?

When investigating PRPS1 phosphorylation during cell cycle progression, include these essential controls:

• Cell synchronization verification:

  • Confirm cell cycle phase by flow cytometry

  • Use phase-specific markers (e.g., cyclins) to validate synchronization

• Phosphorylation site mutants:

  • Generate phospho-deficient (S103A) and phospho-mimetic (S103D/E) PRPS1 mutants

  • Use as controls to validate phospho-specific antibody specificity

  • Compare enzymatic activity between wild-type and mutant PRPS1

• Kinase inhibition controls:

  • Use CDK1 inhibitors to prevent PRPS1 phosphorylation

  • Include kinase-dead CDK1 mutants as negative controls for in vitro assays

• Dephosphorylation controls:

  • Treat samples with phosphatases to remove phosphorylation

  • Verify antibody specificity by loss of signal after phosphatase treatment

How can non-specific binding be minimized in PRPS1 Western blotting?

When encountering non-specific binding issues with PRPS1 antibodies in Western blotting:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Adjust blocking time and temperature

  • Consider using specialized blocking buffers for phospho-specific antibodies

• Antibody dilution optimization:

  • Test a range of dilutions around the recommended 1:1000

  • Optimize primary antibody incubation time and temperature

  • Consider using antibody dilution buffers containing low detergent concentrations

• Washing protocol enhancement:

  • Increase number and duration of wash steps

  • Use TBS-T with optimized Tween-20 concentration

  • Consider adding low concentrations of SDS to wash buffers for stubborn background

• Sample preparation considerations:

  • Include phosphatase inhibitors when detecting phosphorylated PRPS1

  • Use fresh samples to minimize protein degradation

  • Optimize protein loading to avoid oversaturation

What factors influence accurate measurement of PRPS1 enzymatic activity?

Several critical factors can affect PRPS1 activity measurements:

• Enzyme stability considerations:

  • Maintain samples at 4°C during preparation

  • Include protease inhibitors to prevent degradation

  • Minimize time between sample preparation and activity measurement

• Reaction conditions optimization:

  • Verify pH optimum (7.4 is standard but may vary by experimental system)

  • Ensure Mg²⁺ concentration is optimal (5 mM standard)

  • Confirm substrate concentrations are not limiting

• Detection method sensitivity:

  • Calibrate HPLC detection system with AMP standards

  • Ensure linear detection range encompasses expected activity levels

  • Consider alternative detection methods for low abundance samples

• Normalization strategies:

  • Normalize to total PRPS1 protein amount

  • Ensure purification efficiency is consistent between samples

  • Use internal standards for multi-sample comparisons

How can PRPS1 antibodies be used to investigate metabolic reprogramming in cancer?

PRPS1 antibodies can serve as valuable tools in studying cancer metabolic reprogramming:

• Metabolic pathway analysis:

  • Use PRPS1 antibodies to assess enzyme levels in glycolysis vs. PPP utilization

  • Combine with metabolic flux analysis to correlate PRPS1 expression with pathway activity

  • Co-immunoprecipitate PRPS1 with other metabolic enzymes to identify regulatory complexes

• Therapeutic response monitoring:

  • Track PRPS1 phosphorylation status before and after treatment with metabolic inhibitors

  • Correlate PRPS1 activity with resistance to nucleoside analog therapies

  • Use phospho-PRPS1 (S103) as a biomarker for cell cycle-targeted therapy efficacy

• Spatial metabolic heterogeneity:

  • Apply immunohistochemistry with PRPS1 antibodies on tissue sections

  • Map PRPS1 expression/phosphorylation relative to hypoxic or nutrient-deprived regions

  • Correlate with markers of proliferation and metabolic stress

What methodological approaches can assess PRPS1 regulation across different cellular compartments?

PRPS1 may be regulated differently across cellular compartments. To investigate this:

• Subcellular fractionation coupled with immunoblotting:

  • Separate nuclear, cytoplasmic, and mitochondrial fractions

  • Detect PRPS1 and phospho-PRPS1 in each fraction

  • Normalize to compartment-specific markers

• Immunofluorescence microscopy:

  • Use validated PRPS1 antibodies for cellular localization

  • Co-stain with compartment markers and cell cycle indicators

  • Perform quantitative image analysis to assess spatial distribution changes

• Proximity ligation assay (PLA):

  • Detect interactions between PRPS1 and compartment-specific regulatory proteins

  • Visualize changes in interaction patterns during cell cycle progression

  • Quantify interaction differences between normal and cancer cells

• APEX2-based proximity labeling:

  • Fuse PRPS1 with APEX2 enzyme

  • Perform biotinylation of proximal proteins in specific compartments

  • Identify compartment-specific PRPS1 interactors via mass spectrometry