PAPSS2 Antibody

What is PAPSS2 and why is it significant in research?

PAPSS2 is a bifunctional enzyme with both ATP sulfurylase and APS kinase activities that mediates two critical steps in the sulfate activation pathway. The first step involves transferring a sulfate group to ATP to yield adenosine 5'-phosphosulfate (APS), while the second step transfers a phosphate group from ATP to APS, yielding 3'-phosphoadenylylsulfate (PAPS) . In mammals, PAPS serves as the sole source of sulfate for sulfotransferases, while APS appears to function primarily as an intermediate in the sulfate-activation pathway . PAPSS2 plays an essential role in skeletogenesis during postnatal growth, with gene defects causing Pakistani type spondyloepimetaphyseal dysplasia . This protein's involvement in critical biological processes makes PAPSS2 antibodies valuable tools for studying sulfation-dependent pathways in development, disease, and metabolism.

Commercially available PAPSS2 antibodies target different regions of the protein, which can affect their utility in specific applications. Some antibodies recognize epitopes within the N-terminal region (amino acids 1-250) , while others target the C-terminal portion (amino acid 300 to C-terminus) . This distinction is particularly important when:

• Studying truncated protein variants or alternatively spliced isoforms

• Investigating protein complexes where certain domains may be masked

• Examining post-translational modifications in specific regions

• Developing detection strategies for differential expression of PAPSS2 isoforms

Two alternatively spliced transcript variants that encode different isoforms have been described for the PAPSS2 gene . Therefore, when selecting an antibody, researchers should consider which protein domains they need to detect and verify the immunogen sequence to ensure the antibody will recognize their protein of interest.

What are the optimal storage conditions for preserving PAPSS2 antibody activity?

Proper storage is critical for maintaining antibody functionality across extended research timelines. Most PAPSS2 antibodies are supplied in a buffer containing glycerol (typically 50%) and should be stored at -20°C, where they remain stable for approximately 12 months after shipment . To maximize antibody shelf life and performance:

• Avoid repeated freeze-thaw cycles, which can degrade antibody quality and reduce binding efficiency

• Store in manufacturer-recommended buffer conditions (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3)

• Follow manufacturer guidelines regarding aliquoting - some suppliers specifically note that aliquoting is unnecessary or not recommended for -20°C storage

• Upon receipt of antibodies shipped with ice packs, immediately transfer to recommended storage temperature

Extended storage stability can be achieved by maintaining consistent temperature and minimizing exposure to light, heat, and contaminants.

What controls should be incorporated when validating PAPSS2 antibody specificity?

Rigorous validation of PAPSS2 antibodies is essential for generating reliable research data. A comprehensive validation approach should include:

• Positive tissue controls: Human liver tissue and HepG2 cells consistently demonstrate detectable PAPSS2 expression and serve as reliable positive controls

• Negative controls:

  • Primary antibody omission control

  • Isotype-matched irrelevant antibody control (rabbit IgG)

• Knockdown/knockout validation: When possible, PAPSS2 siRNA or CRISPR-edited cell lines provide definitive specificity controls

• Recombinant protein competition: Pre-absorption with immunogen fusion protein can confirm binding specificity

• Multiple antibody verification: Using antibodies targeting different epitopes of PAPSS2 to confirm consistent detection patterns

Documenting these validation steps significantly improves confidence in experimental findings and should be included in methodology sections of publications.

How can researchers optimize Western blot protocols for PAPSS2 detection?

Detecting PAPSS2 in Western blot applications requires specific considerations to achieve optimal signal and specificity. PAPSS2 has a calculated molecular weight of approximately 70 kDa, which corresponds to the observed molecular weight in SDS-PAGE . For optimal PAPSS2 detection:

• Sample preparation:

  • Use RIPA or similar lysis buffers with protease inhibitor cocktails

  • Sonication may improve extraction for membrane-associated fractions

• Gel selection and separation:

  • 7.5% SDS-PAGE provides optimal resolution for the 70 kDa PAPSS2 protein

  • Load 30 μg of whole cell lysate for adequate detection

• Transfer and blocking:

  • Standard PVDF or nitrocellulose membranes are suitable

  • Block with 5% non-fat dry milk or BSA in TBST

• Antibody incubation:

  • Primary antibody dilution ranges from 1:500-1:2400, with 1:1000 being commonly effective

  • Overnight incubation at 4°C generally provides optimal results

• Detection system:

  • HRP-conjugated secondary antibodies with enhanced chemiluminescence work effectively

  • For quantitative analysis, fluorescently-labeled secondary antibodies may offer advantages

Following these methodological considerations will maximize the likelihood of specific PAPSS2 detection in Western blot applications.

What are common challenges in IHC applications of PAPSS2 antibodies and how can they be addressed?

Immunohistochemical detection of PAPSS2 may present several technical challenges. Based on validated protocols, researchers can implement the following strategies to overcome common issues:

• Weak or inconsistent staining:

  • Optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 often works well)

  • Adjust antibody concentration within the recommended range (1:50-1:300)

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

  • Ensure tissue fixation is appropriate (overfixation can mask epitopes)

• High background:

  • Increase blocking duration with 5-10% normal serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Optimize secondary antibody dilution

  • Include an endogenous peroxidase quenching step (3% H₂O₂)

• False-positive results:

  • Include appropriate negative controls in each experiment

  • Validate staining patterns against known PAPSS2 expression profiles

  • Verify specificity with alternative detection methods

PAPSS2 antibodies have been successfully validated in human liver cancer and cervical cancer tissues , making these useful positive controls for method optimization.

When should researchers consider using alternative detection methods to complement PAPSS2 antibody-based assays?

While antibody-based detection offers many advantages, complementary approaches can strengthen research findings, particularly in the following scenarios:

• When examining functional activity rather than protein levels:

  • Enzyme activity assays measuring ATP sulfurylase or APS kinase activity

  • Metabolite analysis of PAPS or downstream sulfated compounds

• When investigating gene expression patterns:

  • RT-qPCR for PAPSS2 mRNA quantification

  • RNA-seq for comprehensive transcriptomic profiling

  • In situ hybridization for localized expression analysis

• When studying genetic variations:

  • PCR-based genotyping for known PAPSS2 mutations

  • Sequencing to identify novel variants

• When antibody cross-reactivity is a concern:

  • Mass spectrometry-based protein identification

  • Recombinant expression with epitope tags for tracking

Integrating multiple methodological approaches provides more robust evidence and can address limitations inherent to any single detection method.

How can PAPSS2 antibodies contribute to understanding disease mechanisms in skeletal disorders?

PAPSS2 plays a critical role in skeletogenesis, and defects in this gene cause the Pakistani type of spondyloepimetaphyseal dysplasia . Researchers investigating skeletal disorders can leverage PAPSS2 antibodies in several sophisticated applications:

• Spatial-temporal expression analysis:

  • Immunohistochemical mapping of PAPSS2 expression in growth plate cartilage during different developmental stages

  • Co-localization studies with chondrogenic markers to understand developmental regulation

• Mechanistic investigations:

  • Immunoprecipitation to identify PAPSS2 binding partners in chondrocytes

  • Phosphorylation status analysis using phospho-specific antibodies to examine regulatory mechanisms

  • Subcellular localization studies using fractionation followed by Western blotting

• Disease model characterization:

  • Comparative analysis of PAPSS2 expression and localization in normal versus pathological tissues

  • Correlation of PAPSS2 levels with severity of cartilage abnormalities

• Therapeutic development:

  • Monitoring PAPSS2 expression in response to experimental treatments

  • Validating gene therapy approaches targeting PAPSS2 deficiency

PAPSS2 antibodies have been instrumental in studies demonstrating its role in osteoarthritis and cartilage maintenance , highlighting their value in skeletal research.

What strategies can researchers employ to investigate PAPSS2's role in cancer biology?

PAPSS2 has been implicated in various cancer types, making it an interesting subject for oncology research. Advanced applications of PAPSS2 antibodies in cancer research include:

• Expression profiling across cancer types:

  • Tissue microarray analysis to correlate PAPSS2 expression with clinical outcomes

  • Comparative studies between primary tumors and metastatic lesions

• Signaling pathway analysis:

  • Phosphoproteomics combined with PAPSS2 immunoprecipitation to identify cancer-specific modifications

  • Investigation of PAPSS2-dependent sulfation in tumor microenvironment

• Functional studies:

  • Immunofluorescence to track subcellular localization changes during epithelial-mesenchymal transition

  • Chromatin immunoprecipitation (ChIP) combined with PAPSS2 antibodies to investigate potential nuclear functions

• Biomarker development:

  • Multiplex IHC panels including PAPSS2 for tumor classification

  • Circulating tumor cell analysis with PAPSS2 detection

PAPSS2 antibodies have been validated in human liver cancer and cervical cancer tissues , providing a foundation for expanding research into additional cancer types.

How does PAPSS2 function differ from PAPSS1, and what methodological approaches can distinguish between them?

While PAPSS1 and PAPSS2 share functional similarities as bifunctional enzymes in the sulfate activation pathway, they differ in tissue distribution and regulatory mechanisms. Researchers can employ several strategies to investigate their distinct roles:

• Antibody-based differentiation:

  • Select isoform-specific antibodies validated for lack of cross-reactivity

  • Use epitope-mapped antibodies targeting non-conserved regions

  • Perform parallel detection of both isoforms in the same samples

• Expression pattern analysis:

  • Compare PAPSS1 and PAPSS2 distribution across tissues using validated antibodies

  • Examine differential regulation under various physiological stimuli

• Functional comparison:

  • Conduct selective knockdown studies followed by antibody-based detection

  • Analyze compensatory expression changes when one isoform is depleted

• Structural investigations:

  • Use antibodies recognizing specific conformational states

  • Combine with structural biology approaches for comprehensive understanding

What methodological approaches combine PAPSS2 antibody detection with systems biology techniques?

Integrating PAPSS2 antibody-based detection with systems biology approaches can provide comprehensive insights into sulfation pathways. Advanced integrative strategies include:

• Multi-omics integration:

  • Correlate PAPSS2 protein levels (detected by antibodies) with transcriptomic profiles

  • Map PAPSS2 expression onto metabolomic data of sulfated compounds

  • Integrate with epigenomic data to understand regulatory mechanisms

• Network analysis:

  • Use co-immunoprecipitation with PAPSS2 antibodies followed by mass spectrometry to map protein interaction networks

  • Validate key interactions with proximity ligation assays

• Spatial biology approaches:

  • Multiplex immunofluorescence to map PAPSS2 in relation to other pathway components

  • Spatial transcriptomics correlated with PAPSS2 protein distribution

• Temporal dynamics:

  • Time-course studies combining PAPSS2 antibody detection with functional readouts

  • Live-cell imaging with fluorescently-tagged antibody derivatives

These integrated approaches provide contextual understanding of PAPSS2 function beyond what can be achieved with single-method investigations.