PRPSAP1 Antibody

What is PRPSAP1 and why is it significant for research?

PRPSAP1 (phosphoribosyl pyrophosphate synthetase-associated protein 1), also known as PAP39, is a 356 amino acid protein with a calculated molecular weight of 39 kDa that plays a negative regulatory role in 5-phosphoribose 1-diphosphate synthesis . The protein binds catalytic subunits of the ribose-phosphate diphosphokinase enzyme complex (PRPS1 and PRPS2), forming a regulatory complex essential for nucleotide metabolism . PRPSAP1's regulation is critical for cellular proliferation and metabolism, making it a significant target for studies related to cancer and metabolic disorders . The gene encoding PRPSAP1 is located on human chromosome 17q25.1, and its evolutionary conservation highlights its biological significance across species .

What types of PRPSAP1 antibodies are currently available for research?

Several types of PRPSAP1 antibodies are available for research applications:

Most antibodies target specific epitopes within the PRPSAP1 protein, including N-terminal, middle region, and C-terminal domains . The majority of these antibodies have been validated through multiple applications and sample types, providing researchers with reliable tools for PRPSAP1 detection and characterization .

How should I select the appropriate PRPSAP1 antibody for my specific application?

Selection of the appropriate PRPSAP1 antibody depends on several critical factors:

• Experimental application: Different antibodies are optimized for specific applications. For Western blotting, antibodies like 16790-1-AP have been extensively validated at dilutions of 1:500-1:1000 . For immunohistochemistry, consider antibodies validated at 1:50-1:500 dilutions with demonstrated reactivity in tissues of interest .

• Species reactivity: Ensure the antibody recognizes PRPSAP1 in your species of interest. Many antibodies react with human, mouse, and rat PRPSAP1, but cross-reactivity with other species varies .

• Epitope specificity: For detecting specific domains, select antibodies targeting relevant regions. N-terminal antibodies like those recognizing amino acids 30-110 are available for specific structural studies .

• Clonality requirements: Polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide higher specificity for single epitopes .

• Conjugation needs: Consider whether your experiment requires direct detection (conjugated antibodies) or indirect detection (unconjugated antibodies with secondary detection) .

Always review validation data, including Western blot images, IHC staining patterns, and positive control samples before selection .

What validation procedures should be performed before using a PRPSAP1 antibody in critical experiments?

Before using PRPSAP1 antibodies in critical experiments, comprehensive validation is essential:

• Positive control testing: Validate antibody performance using tissues or cell lines with known PRPSAP1 expression. Human testis tissue and HeLa cells have been successfully used as positive controls for Western blotting .

• Dilution optimization: Perform titration experiments to determine optimal working concentrations. For example, Western blot applications typically require 1:500-1:2,000 dilutions, while IHC applications may require 1:50-1:500 dilutions .

• Specificity confirmation: Verify a single band of appropriate molecular weight (approximately 39 kDa) in Western blot applications . For IHC applications, confirm specific staining patterns in positive control tissues like human liver cancer or cervical cancer tissues .

• Negative controls: Include samples lacking PRPSAP1 expression or use blocking peptides to confirm specificity of the signal .

• Cross-reactivity assessment: If working across species, verify cross-reactivity with the target protein in each species of interest through comparative analysis .

• Batch consistency verification: When receiving new lots, compare performance with previously validated lots using the same experimental conditions and samples .

What are the optimized protocols for using PRPSAP1 antibodies in Western blotting?

For optimal Western blot results with PRPSAP1 antibodies:

• Sample preparation:

  • Use fresh tissue or cell lysates in appropriate lysis buffer

  • Human testis tissue and HeLa cells work well as positive controls

  • Load 30 μg of total protein per lane for cell lysates

• Gel electrophoresis and transfer:

  • 10% SDS-PAGE is recommended for optimal separation

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

• Antibody incubation:

  • Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Primary antibody: Dilute PRPSAP1 antibody at 1:500-1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Secondary antibody: Anti-rabbit or anti-mouse HRP-conjugated antibody at 1:2000-1:5000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) reagents for detection

  • Expected band size: 39 kDa

• Controls:

  • Include positive control samples (HeLa, HepG2 cells)

  • Include negative control (secondary antibody only)

The antibody should detect a clean band at approximately 39 kDa corresponding to PRPSAP1 .

How should PRPSAP1 antibodies be used in immunohistochemistry applications?

For successful IHC applications with PRPSAP1 antibodies:

• Tissue preparation:

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

  • Human liver cancer and cervical cancer tissues have been validated as positive controls

• Antigen retrieval:

  • Recommended: TE buffer, pH 9.0

  • Alternative: Citrate buffer, pH 6.0

  • Heat-induced epitope retrieval (HIER) at 95-100°C for 15-20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5-10% normal serum

  • Primary antibody dilution: 1:50-1:500 depending on specific antibody

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

• Detection system:

  • Use an appropriate detection system (HRP/DAB or AP/Red)

  • Counterstain with hematoxylin

  • Mount with permanent mounting medium

• Controls:

  • Include positive control tissues with known PRPSAP1 expression

  • Include negative control (omit primary antibody)

  • For specific antibodies, validated positive samples include SW480 xenograft tissues

Optimal antibody dilution should be determined empirically for each tissue type and fixation method .

What are the recommended protocols for immunoprecipitation with PRPSAP1 antibodies?

For effective immunoprecipitation of PRPSAP1:

• Sample preparation:

  • Prepare cell/tissue lysate in a non-denaturing lysis buffer containing protease inhibitors

  • Mouse testis tissue has been validated for successful IP applications

  • Clear lysate by centrifugation (14,000 × g, 10 minutes at 4°C)

• Pre-clearing (optional):

  • Incubate lysate with protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 0.5-4.0 μg of PRPSAP1 antibody to 1.0-3.0 mg of total protein lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 30-50 μl of protein A/G beads

  • Incubate for 2-4 hours at 4°C with gentle rotation

• Washing and elution:

  • Wash beads 3-5 times with lysis buffer

  • Elute with SDS sample buffer by heating at 95°C for 5 minutes

• Analysis:

  • Analyze by Western blot using a different PRPSAP1 antibody or the same antibody

  • Expected band size: 39 kDa

For successful IP, it is recommended to verify antibody-antigen binding capacity before performing full-scale experiments .

What are common issues encountered in Western blotting with PRPSAP1 antibodies and how can they be resolved?

Common Western blotting issues with PRPSAP1 antibodies and their solutions:

Problem 1: No signal or weak signal

• Potential causes: Insufficient protein, inefficient transfer, degraded antibody, low PRPSAP1 expression

• Solutions:

  • Increase protein loading (30-50 μg recommended)

  • Verify transfer efficiency with Ponceau S staining

  • Use fresh antibody at optimal concentration (1:500-1:1000)

  • Include positive controls (human testis tissue, HeLa cells)

  • Extend primary antibody incubation to overnight at 4°C

  • Use more sensitive detection method (e.g., enhanced ECL)

Problem 2: Multiple bands or non-specific signals

• Potential causes: Cross-reactivity, protein degradation, antibody concentration too high

• Solutions:

  • Optimize blocking conditions (5% milk or BSA)

  • Use freshly prepared samples with protease inhibitors

  • Reduce antibody concentration

  • Perform more stringent washing steps (increase TBST washing time/frequency)

  • Use a different PRPSAP1 antibody targeting a different epitope

Problem 3: High background

• Potential causes: Insufficient blocking, antibody concentration too high, contaminated reagents

• Solutions:

  • Extend blocking time (1-2 hours at room temperature)

  • Dilute primary and secondary antibodies further

  • Use fresh blocking reagents and TBST

  • Add 0.05-0.1% Tween-20 to antibody dilution buffer

  • Increase washing duration and frequency

What technical challenges might arise in immunohistochemistry with PRPSAP1 antibodies and how can they be addressed?

Common IHC challenges with PRPSAP1 antibodies and their solutions:

Problem 1: Weak or no staining

• Potential causes: Insufficient antigen retrieval, low antibody concentration, low PRPSAP1 expression

• Solutions:

  • Optimize antigen retrieval (try TE buffer pH 9.0 as recommended for PRPSAP1)

  • Increase antibody concentration (start with 1:50 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use amplification systems (e.g., tyramide signal amplification)

  • Confirm PRPSAP1 expression in the tissue being examined

Problem 2: High background or non-specific staining

• Potential causes: Inadequate blocking, antibody concentration too high, endogenous enzyme activity

• Solutions:

  • Optimize blocking steps (use 5-10% normal serum from the same species as secondary antibody)

  • Block endogenous peroxidase (3% H₂O₂) and biotin if using biotin-based detection

  • Reduce antibody concentration (try 1:200-1:500 dilution)

  • Include additional washing steps

  • Use different detection system

Problem 3: Inconsistent staining across tissue sections

• Potential causes: Uneven fixation, variable antigen retrieval, tissue drying

• Solutions:

  • Ensure consistent fixation time and processing

  • Perform antigen retrieval in a controlled environment (e.g., pressure cooker)

  • Prevent tissue drying during staining procedure

  • Apply antibody solution evenly and use humidified chamber

  • Consider automated staining platforms for consistency

How can PRPSAP1 antibodies be used in studying protein-protein interactions and complex formation?

PRPSAP1 antibodies can be valuable tools for investigating protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use PRPSAP1 antibodies to immunoprecipitate native protein complexes

  • Analyze co-precipitated proteins (especially PRPS1 and PRPS2) by Western blotting or mass spectrometry

  • Recommended antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • This approach has successfully identified interactions between PRPSAP1 and catalytic subunits of the phosphoribosylpyrophosphate synthetase complex

• Proximity Ligation Assay (PLA):

  • Combine PRPSAP1 antibodies with antibodies against potential interacting partners

  • Detect protein interactions with single-molecule resolution in fixed cells or tissues

  • Requires primary antibodies from different host species (rabbit anti-PRPSAP1 can be paired with mouse antibodies against potential interactors)

• Immunofluorescence co-localization:

  • Use fluorescently labeled PRPSAP1 antibodies (or primary antibody + fluorescent secondary)

  • Co-stain with antibodies against potential interacting proteins

  • Analyze co-localization patterns by confocal microscopy

  • FITC-conjugated PRPSAP1 antibodies are available for direct detection

• Bimolecular Fluorescence Complementation (BiFC):

  • Express PRPSAP1 and potential interacting proteins as fusion constructs

  • Use PRPSAP1 antibodies to confirm expression levels

  • This technique allows visualization of protein interactions in living cells

These approaches can help elucidate PRPSAP1's role in regulating phosphoribosylpyrophosphate production and nucleotide biosynthesis pathways .

What are the considerations for using PRPSAP1 antibodies in studying conformational changes in prion disease research?

PRPSAP1 antibodies can provide insights into conformational changes in proteins, which is particularly relevant for prion disease research:

• Conformational epitope recognition:

  • Some antibodies recognize specific conformational epitopes that may be altered in disease states

  • PrP^Sc-specific antibodies have demonstrated the ability to detect strain-dependent conformational variations in the C-terminus of prion proteins

  • This principle can be applied to study potential conformational changes in PRPSAP1 under different pathological conditions

• Differential epitope accessibility analysis:

  • Use a panel of PRPSAP1 antibodies targeting different epitopes

  • Compare binding patterns under native vs. denatured conditions

  • This approach has revealed strain-specific conformational differences in prion proteins

  • For PRPSAP1, this may reveal functional states related to its regulatory activity

• Enzyme-linked immunosorbent assay (ELISA) applications:

  • Systematic testing of epitope accessibility using indirect ELISA

  • Compare folded (untreated) vs. denatured (GdnHCl-treated) protein conformations

  • Include appropriate controls by omitting primary or secondary antibodies

  • This technique has successfully distinguished different prion strains and could potentially reveal PRPSAP1 conformational states

• Methodological considerations:

  • Careful sample preparation to preserve native protein conformations

  • Use non-denaturing conditions during extraction and analysis

  • Include both native and denatured samples as controls

  • Quantify antibody binding through standardized protocols

Research on prion proteins has shown that conformational antibodies can detect subtle structural differences between strains, providing a model for similar studies with PRPSAP1 .

How can PRPSAP1 antibodies be utilized in CRISPR-based gene regulation studies?

PRPSAP1 antibodies are valuable tools in CRISPR-based gene regulation studies:

• Monitoring gene activation efficiency:

  • CRISPR Activation (CRISPRa) systems are available for PRPSAP1 upregulation

  • PRPSAP1 antibodies can be used to verify increased protein expression following CRISPRa

  • Recommended antibody: PRPSAP1 (A-9): sc-398422 for monitoring gene expression by Western blotting

• Validation of CRISPR knock-out or knock-down:

  • Confirm complete protein depletion in CRISPR knockout models

  • Assess partial reduction in protein levels in CRISPR interference (CRISPRi) approaches

  • Western blotting at 1:500-1:1000 dilution is recommended for detection

• Phenotypic characterization:

  • Use immunohistochemistry or immunofluorescence to examine cellular localization changes

  • Compare PRPSAP1 expression patterns between wild-type and CRISPR-modified cells

  • Analyze tissue-specific effects in CRISPR-modified animal models

  • IHC applications at 1:50-1:500 dilution can detect expression changes

• Protein-protein interaction alterations:

  • Apply Co-IP using PRPSAP1 antibodies to determine how CRISPR modifications affect protein complex formation

  • Investigate changes in PRPSAP1 interactions with PRPS1/PRPS2 following gene editing

  • IP protocol using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein is recommended

• Technical considerations:

  • Include appropriate controls (non-targeting gRNA, wild-type cells)

  • Validate antibody specificity in the context of your CRISPR system

  • Consider temporal aspects of protein depletion/induction when planning experiments

These approaches can help elucidate PRPSAP1's functional role in nucleotide metabolism and cellular proliferation through precise genetic manipulation combined with antibody-based detection .

How can PRPSAP1 antibodies be employed in cancer research studies?

PRPSAP1 antibodies can be valuable tools in cancer research due to the protein's role in nucleotide metabolism and potential implications in cellular proliferation:

• Expression profiling in tumor tissues:

  • IHC analysis of PRPSAP1 expression across cancer types

  • Validated positive samples include human liver cancer and cervical cancer tissues

  • Recommended antibody dilutions: 1:50-1:500 for IHC applications

  • Correlation of expression levels with clinical parameters and patient outcomes

• Metabolic pathway analysis:

  • Western blot detection of PRPSAP1 in cancer cell lines (HeLa, HepG2 validated)

  • Investigation of PRPSAP1's regulatory role in nucleotide synthesis in rapidly proliferating cancer cells

  • Study potential alterations in PRPSAP1-PRPS interactions in transformed cells

  • Recommended antibody dilutions: 1:500-1:1000 for WB applications

• Functional studies:

  • Combine PRPSAP1 antibodies with CRISPR-based approaches to assess functional consequences of expression modulation

  • Investigate effects on cancer cell proliferation, metabolism, and drug response

  • Correlate with changes in nucleotide synthesis pathways

• Therapeutic target validation:

  • Use PRPSAP1 antibodies to verify target engagement in drug development studies

  • Monitor protein levels following treatment with metabolism-targeting agents

  • Assess potential post-translational modifications in response to therapy

What are the methodological considerations for using PRPSAP1 antibodies in studying metabolic disorders?

When investigating metabolic disorders using PRPSAP1 antibodies, several methodological considerations are important:

• Tissue-specific expression analysis:

  • Different tissues may exhibit variable PRPSAP1 expression levels

  • Validated tissues include human testis, liver cancer, and cervical cancer tissues

  • Mouse heart tissue has also been validated as a positive sample

  • Optimize antibody dilutions for each tissue type (1:50-1:500 for IHC)

• Subcellular localization studies:

  • PRPSAP1 is primarily cytoplasmic

  • Immunofluorescence can detect potential subcellular redistribution in metabolic disorders

  • Use FITC-conjugated PRPSAP1 antibodies for direct detection

  • Compare localization patterns between normal and diseased tissues

• Protein-protein interaction alterations:

  • Metabolic disorders may affect PRPSAP1 interactions with PRPS1/PRPS2

  • Co-immunoprecipitation protocols using 0.5-4.0 μg antibody for 1.0-3.0 mg lysate

  • Western blot analysis of complexes at 1:500-1:1000 dilution

  • Consider native vs. denatured protein analysis to capture conformational changes

• Post-translational modifications:

  • Altered metabolism may induce PTMs on PRPSAP1

  • Western blotting can detect mobility shifts indicating modifications

  • Consider phosphorylation-specific antibodies if available

  • Use 10% SDS-PAGE for optimal separation

• Sample handling considerations:

  • Rapid sample processing to prevent degradation of metabolic enzymes

  • Inclusion of phosphatase and protease inhibitors in lysis buffers

  • Storage at -20°C with 50% glycerol for antibody stability

  • Consider flash-freezing tissues to preserve metabolic state

These methodological approaches can help elucidate PRPSAP1's role in metabolic regulation and potential implications in disorders affecting nucleotide metabolism pathways .