PRPS2 Antibody, Biotin conjugated

What is PRPS2 and why is it a significant research target?

PRPS2 (Phosphoribosyl pyrophosphate synthetase 2) is a rate-limiting enzyme that plays a crucial role in purine and pyrimidine nucleotide synthesis. This enzyme catalyzes the synthesis of phosphoribosyl pyrophosphate (PRPP), which is essential for nucleotide metabolism . PRPS2 has gained significant research interest due to its involvement in several biological processes:

• Male reproductive health: PRPS2 depletion has been linked to hypospermatogenesis and increased apoptosis of spermatogenic cells

• Cell proliferation pathways: As a key enzyme in nucleotide synthesis, it influences cellular proliferation mechanisms

• Differential roles from other PRPS isoforms: Unlike PRPS1, which plays a role in normal cell nucleotide metabolism, PRPS2 has been associated with tumor formation and development

Understanding PRPS2 function provides insights into both normal physiological processes and pathological conditions, making it a valuable research target.

How does biotin conjugation enhance antibody functionality in PRPS2 research?

Biotin conjugation significantly enhances antibody functionality through several mechanisms:

• Signal amplification: Biotin-labeled antibodies, when combined with streptavidin- or avidin-based conjugates, allow for signal amplification of lowly expressed proteins

• Versatility across applications: Biotin-conjugated antibodies can be used in multiple applications including western blotting, ELISA, immunohistochemistry, immunocytochemistry, immunofluorescence, and flow cytometry

• Bridged detection systems: The Bridged Avidin-Biotin (BRAB) method enables complex detection systems where antigens are "sandwiched" between capture antibodies and biotin-labeled antibodies

• Enhanced sensitivity: The high-affinity biotin-(strept)avidin interaction (Kd ≈ 10^-15 M) provides exceptional sensitivity for detecting proteins expressed at low levels

This conjugation strategy is particularly valuable for PRPS2 research where precise localization and quantification are essential for understanding its role in cellular processes.

What is the optimal protocol for antibody biotinylation when working with PRPS2 antibodies?

The optimal protocol for biotinylating PRPS2 antibodies involves using long-chain biotin NHS ester (LCB-NHS) to ensure maximal activity and specificity:

Standard Laboratory Protocol:

• Prepare LCB-NHS solution in DMSO at 6 mg/ml (6.5 mM)

• Add 10 µl of LCB-NHS solution to 1 ml of 2 mg/ml PRPS2 antibody

• Incubate at room temperature for 50 minutes to allow complete conjugation

• Terminate the reaction by adding 0.5 ml of 1M Tris-HCl, pH 8.0

• Dialyze for 24-48 hours against 1X PBS to remove unreacted biotin

• Add 0.1% (w/v) NaN₃ as a preservative if necessary

Critical considerations:

• Maintain a molar ratio of biotin:antibody between 4:1 and 8:1 to achieve optimal labeling without compromising antibody binding

• Store conjugated antibodies in light-protected vials or covered with aluminum foil

• For long-term storage (>12 months), dilute conjugates with up to 50% glycerol and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles as they compromise both enzyme activity and antibody binding

This protocol ensures efficient biotinylation while preserving the functional integrity of the PRPS2 antibody.

How should researchers optimize western blotting conditions for biotin-conjugated PRPS2 antibodies?

Optimizing western blotting conditions for biotin-conjugated PRPS2 antibodies requires careful attention to several parameters:

Sample preparation and electrophoresis:

• PRPS2 has an observed molecular weight of 30-34 kDa ; use 10% SDS-PAGE gels for optimal resolution

• Include positive control samples such as A375 cells, which have demonstrated positive western blot results with PRPS2 antibodies

• Load 20-30 μg of total protein per lane for optimal detection

Blotting and detection optimization:

• Transfer proteins to nitrocellulose or PVDF membranes using standard protocols

• Block with 5% non-fat milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20)

• Critical step: Dilute biotin-conjugated primary PRPS2 antibodies at 1:500-1:2000 ratio in blocking buffer

• Incubate membrane overnight at 4°C with gentle rocking

• Wash thoroughly with TBST (3-5 times, 5 minutes each)

• Incubate with streptavidin-HRP (1:5000-1:10000) for 1 hour at room temperature

• Wash thoroughly with TBST (3-5 times, 5 minutes each)

• Develop using chemiluminescence substrate and image

Troubleshooting considerations:

• If background is high, increase washing steps and/or decrease streptavidin-HRP concentration

• If signal is weak, increase antibody concentration or extend incubation time

• Pre-absorb antibodies with blocking proteins if non-specific binding occurs

This optimized protocol accounts for the specific characteristics of biotin-conjugated PRPS2 antibodies, ensuring consistent and reliable results.

How can researchers effectively use biotin-conjugated PRPS2 antibodies in multiplex immunoassays?

Biotin-conjugated PRPS2 antibodies offer significant advantages in multiplex immunoassays through strategic implementation:

Cytometric Bead Array (CBA) Applications:

• Utilize matched antibody pairs such as 85090-3-PBS (capture) and 85090-1-PBS (detection) for PRPS2

• Conjugate detection antibodies with biotin following established protocols

• Employ streptavidin-fluorophore conjugates with distinct emission spectra for multiplexing

• Include appropriate controls to establish assay specificity and sensitivity

Multiplex Imaging Strategies:

• Design sequential staining protocols to prevent cross-reactivity between antibodies

• Use biotin-conjugated PRPS2 antibodies at optimized dilutions (1:200-1:800 for IF/ICC)

• Employ orthogonal detection systems (e.g., directly labeled antibodies for other targets)

• Conduct spectral unmixing if using fluorophores with overlapping emission profiles

Critical Considerations:

• Validate antibody specificity in single-target experiments before multiplexing

• Optimize signal-to-noise ratios for each target in the multiplex panel

• Consider the spatial distribution of targets when designing multiplexing strategies

• Implement computational analysis for quantitative evaluation of multiplexed signals

These approaches enable researchers to simultaneously analyze PRPS2 alongside other biomarkers of interest, providing more comprehensive insights into biological processes while conserving valuable samples.

What are the key considerations for using biotin-conjugated PRPS2 antibodies in studying spermatogenesis and male infertility?

When investigating PRPS2's role in spermatogenesis and male infertility, researchers should consider several critical factors:

Experimental Design Considerations:

• Include appropriate mouse models of hypospermatogenesis as established reference systems

• Compare PRPS2 expression levels across developmental stages (4-10 weeks) to capture temporal patterns

• Incorporate cellular models such as GC1-spg (spermatogonial cell line) and GC2-spg (spermatocyte cell line) for in vitro studies

• Design experiments to assess both PRPS2 knockdown and overexpression effects

Key Methodological Approaches:

• Flow cytometry analysis: Quantify apoptotic rates in spermatogenic cells using annexin V-FITC/propidium iodide staining

• Immunofluorescence staining: Analyze PRPS2 and E2F1 colocalization in testicular tissue

• Western blot analysis: Evaluate expression of apoptotic markers (Caspase 6, Caspase 9) in relation to PRPS2 levels

• Gene expression profiling: Examine downstream effects of PRPS2 modulation on apoptosis-related genes

Data Interpretation Framework:

• Consider that PRPS2 depletion increases apoptotic rates in spermatogenic cells (~16.1% in GC1/shPRPS2 vs. 2.8% in controls)

• Recognize that PRPS2 regulates E2F1, which in turn modulates the P53/Bcl-xl/Bcl-2/Caspase 6/Caspase 9 apoptosis pathway

• Note that PRPS2 expression progressively increases during normal testicular development

This comprehensive approach allows researchers to establish mechanistic links between PRPS2 function and male reproductive disorders, potentially identifying therapeutic targets or diagnostic markers.

How can researchers validate the specificity of biotin-conjugated PRPS2 antibodies?

Validating the specificity of biotin-conjugated PRPS2 antibodies requires a multi-faceted approach:

Recommended Validation Strategy:

• Knockout/Knockdown Controls:

  • Compare staining patterns between wild-type samples and those with PRPS2 knockdown

  • Use siRNA or shRNA to specifically reduce PRPS2 expression in cell lines (e.g., GC1-spg, GC2-spg)

  • Expected result: Significant reduction in signal intensity corresponding to PRPS2 depletion

• Cross-Reactivity Assessment:

  • Test the antibody against recombinant PRPS1 and PRPS2 proteins to confirm isoform specificity

  • Evaluate antibody performance in tissues known to express different levels of PRPS2

  • Compare reactivity patterns across multiple species if using in comparative studies

• Orthogonal Detection Methods:

  • Confirm PRPS2 expression using multiple antibodies targeting different epitopes

  • Verify protein expression using gene expression analysis (qRT-PCR)

  • Compare results from different applications (WB, IHC, IF) for consistency

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (e.g., catalog # AAP41510 for ARP41510_T100-Biotin)

  • Perform parallel experiments with blocked and unblocked antibody

  • Expected result: Specific signals should be eliminated or significantly reduced

Data Analysis Considerations:

• Document antibody validation in publications following guidelines from the International Working Group for Antibody Validation

• Include appropriate positive controls (A375 cells, mouse spleen tissue)

• Report quantitative metrics of specificity (signal-to-noise ratio, coefficient of variation)

This systematic validation approach ensures that experimental findings accurately reflect PRPS2 biology rather than antibody artifacts.

What are common technical challenges when using biotin-conjugated PRPS2 antibodies and how can they be addressed?

Researchers working with biotin-conjugated PRPS2 antibodies may encounter several technical challenges, each requiring specific troubleshooting approaches:

High Background in Immunoassays

Inconsistent Signal Intensity

Cross-Reactivity Issues

Signal Amplification Control

Implementing these systematic troubleshooting approaches will help researchers overcome technical challenges and obtain reliable, reproducible results when working with biotin-conjugated PRPS2 antibodies.

How can biotin-conjugated PRPS2 antibodies contribute to understanding the E2F1-mediated apoptosis pathway in spermatogenic cells?

Biotin-conjugated PRPS2 antibodies provide powerful tools for dissecting the E2F1-mediated apoptosis pathway in spermatogenic cells:

Mechanistic Insights from Current Research:

• PRPS2 and E2F1 are colocalized in GC1 and GC2 cells and normal testicular tissue, suggesting a functional relationship

• PRPS2 downregulation decreases E2F1 expression, establishing a regulatory connection

• E2F1 overexpression can rescue spermatogenic cells from apoptosis induced by PRPS2 depletion

• The apoptotic pathway involves P53/Bcl-xl/Bcl-2/Caspase 6/Caspase 9 regulation downstream of E2F1

Advanced Research Applications:

• Co-Immunoprecipitation (Co-IP):

  • Use biotin-conjugated PRPS2 antibodies to pull down protein complexes

  • Analyze interactions between PRPS2, E2F1, and other regulatory proteins

  • Map the temporal dynamics of these interactions during spermatogenesis

• Chromatin Immunoprecipitation (ChIP):

  • Investigate whether PRPS2 directly or indirectly regulates E2F1 at the transcriptional level

  • Map genomic binding sites using ChIP-seq approaches

  • Correlate binding patterns with gene expression changes

• Proximity Ligation Assays (PLA):

  • Visualize and quantify PRPS2-E2F1 interactions in situ

  • Compare interaction patterns between normal and pathological samples

  • Assess how these interactions change in response to experimental manipulations

• Live-Cell Imaging:

  • Develop systems using biotin-conjugated antibody fragments for live-cell imaging

  • Track PRPS2 and E2F1 dynamics during apoptosis induction

  • Correlate protein redistribution with cellular phenotypes

Potential Research Questions:

• Does PRPS2 regulate E2F1 directly through protein-protein interactions or indirectly through metabolic effects on nucleotide synthesis?

• How do PRPS2-E2F1 interactions differ between normal spermatogenesis and hypospermatogenesis?

• Can targeting the PRPS2-E2F1 axis provide therapeutic opportunities for male infertility?

These approaches using biotin-conjugated PRPS2 antibodies would significantly advance our understanding of this critical pathway in male reproductive biology.

What emerging applications for biotin-conjugated PRPS2 antibodies show promise in reproductive medicine and cancer research?

Biotin-conjugated PRPS2 antibodies are enabling innovative applications in both reproductive medicine and cancer research:

Emerging Applications in Reproductive Medicine:

• Diagnostic Biomarker Development:

  • PRPS2 depletion is associated with hypospermatogenesis, suggesting potential as a diagnostic marker

  • Biotin-conjugated antibodies could enable multiplexed screening of testicular biopsies

  • Quantitative analysis of PRPS2 expression patterns may help classify male infertility subtypes

• Single-Cell Profiling of Testicular Tissue:

  • Biotin-conjugated PRPS2 antibodies can be integrated into mass cytometry or imaging mass cytometry

  • This allows comprehensive phenotyping of heterogeneous testicular cell populations

  • Correlation of PRPS2 expression with cell differentiation states provides insights into spermatogenesis

• Therapeutic Target Validation:

  • PRPS2 modulation affects the survival of spermatogenic cells through E2F1 and apoptotic pathways

  • Biotin-conjugated antibodies enable high-throughput screening for compounds that modulate PRPS2 function

  • This approach could identify potential treatments for specific forms of male infertility

Promising Applications in Cancer Research:

• Differential Metabolic Dependencies:

  • PRPS2, but not PRPS1, is associated with tumor formation and development

  • Biotin-conjugated antibodies can help map PRPS2 expression across cancer types

  • This may reveal cancer-specific vulnerabilities in nucleotide metabolism pathways

• Therapeutic Resistance Mechanisms:

  • PRPS2's role in nucleotide synthesis suggests potential involvement in resistance to antimetabolite therapies

  • Multiplex imaging with biotin-conjugated PRPS2 antibodies can reveal spatial relationships with drug resistance markers

  • This could inform combination therapy approaches targeting both PRPS2 and complementary pathways

• Cell Death Pathway Analysis:

  • PRPS2 regulation of E2F1 and downstream apoptotic pathways has implications beyond reproductive biology

  • Biotin-conjugated PRPS2 antibodies enable dynamic monitoring of these pathways in response to therapeutic interventions

  • This approach could identify synthetic lethal interactions for cancer treatment

Future Research Directions:

• Development of conditional PRPS2 knockout models to further elucidate tissue-specific functions

• Investigation of PRPS2's role in cancer stem cell maintenance and therapy resistance

• Exploration of PRPS2 as a target for male contraceptive development

These emerging applications highlight the versatility of biotin-conjugated PRPS2 antibodies across multiple fields of biomedical research.

What are the recommended storage and handling procedures to maintain optimal activity of biotin-conjugated PRPS2 antibodies?

Proper storage and handling are critical for maintaining the activity and specificity of biotin-conjugated PRPS2 antibodies:

Storage Conditions by Duration:

Critical Handling Procedures:

• Light Protection:

  • Store all biotin-conjugated antibodies in light-protected vials or wrapped in aluminum foil

  • Minimize exposure to light during experimental procedures

  • Light exposure can reduce biotin activity and increase background

• Aliquoting Strategy:

  • Upon receipt, divide antibody into single-use aliquots

  • For antibodies with 0.02% sodium azide and 50% glycerol (pH 7.2), aliquoting is unnecessary for -20°C storage

  • For other formulations, aliquoting prevents repeated freeze-thaw cycles

• Thawing Protocol:

  • Thaw frozen antibodies completely at 4°C or on ice

  • Mix gently by inversion, avoid vortexing which can denature antibodies

  • Briefly centrifuge to collect contents at the bottom of the tube

• Working Solution Preparation:

  • Dilute antibodies in fresh, cold buffer immediately before use

  • For applications requiring lower background, prepare working solutions with fresh buffer containing 0.1-0.5% BSA

  • Use polypropylene tubes to prevent antibody adherence to container walls

Recommended Storage Buffers:

Quality Control Measures:

• Before each use, check for visible precipitation or unusual color changes

• Periodically test antibody performance using positive controls (e.g., A375 cells for PRPS2)

• Document lot number, receipt date, and thawing dates for troubleshooting

Following these storage and handling recommendations will help ensure consistent performance and extend the useful life of biotin-conjugated PRPS2 antibodies.

What reference materials and validation datasets should researchers consult when working with biotin-conjugated PRPS2 antibodies?

Researchers should consult comprehensive reference materials and validation datasets to ensure reliable results with biotin-conjugated PRPS2 antibodies:

Essential Reference Resources:

• Protein Databases and Tools:

  • UniProt ID: P11908 - Contains comprehensive protein information for human PRPS2

  • NCBI Gene ID: 5634 - Provides genomic context and expression data

  • BioGPS - Confirms PRPS2 expression in relevant cell types (e.g., Jurkat cells)

• Antibody Validation Repositories:

  • Antibodypedia - Collates user experiences and validation data

  • Protein Atlas - Provides tissue expression maps for PRPS2

  • CiteAb - Compiles citations where specific antibodies have been used successfully

• Published Validation Images:

  • Western blot validation showing PRPS2 at 30-34 kDa in various cell lines

  • Immunofluorescence images demonstrating subcellular localization patterns

  • Immunohistochemistry results in tissues like human stomach

Recommended Validation Datasets:

Methodological Reference Studies:

• PRPS2 in Male Fertility:

  • Study demonstrating PRPS2 depletion effects on spermatogenic cell apoptosis

  • Experimental protocols for mouse models of hypospermatogenesis

  • Methods for analyzing E2F1-mediated apoptosis pathways

• Biotin Conjugation Techniques:

  • Protocols for antibody biotinylation

  • Studies on the biotin–(strept)avidin system in immunoassays

  • Guidelines for optimizing biotin-streptavidin detection systems

• Advanced Applications:

  • Publications using matched antibody pairs for cytometric bead arrays

  • Studies employing multiplex imaging with biotin-conjugated antibodies

  • Research implementing the Bridged Avidin-Biotin method

Critical Assessment Framework:

• Compare validation data across multiple antibody vendors

• Assess consistency between orthogonal detection methods

• Evaluate reproducibility across independent studies

• Consider context-specific factors (species, tissue type, fixation method)