PIAS2 Antibody

Basic Research Questions

PIAS2 exists in multiple isoforms produced by alternative splicing, which can complicate band interpretation in Western blot analysis:

• Calculated molecular weights: 63 kDa and 69 kDa

• Observed molecular weights: 63-70 kDa range

• Specific isoforms: 68 kDa, 63 kDa, and 45 kDa

The predominant band typically appears at approximately 68 kDa in most cell lines . When analyzing Western blot results, be aware that post-translational modifications, particularly SUMOylation, may cause band shifts. If using recombinant PIAS2 as a positive control, note that the observed molecular weight may differ slightly from endogenous protein .

Which cell lines serve as effective positive controls for PIAS2 antibody validation?

Several cell lines consistently express detectable levels of PIAS2 and are recommended as positive controls:

• HeLa cells: Human cervical epithelial carcinoma cell line shows robust PIAS2 expression

• MCF-7 cells: Human breast cancer cell line demonstrates reliable PIAS2 expression for both WB and IF applications

• K-562 cells: Human chronic myelogenous leukemia cell line exhibits consistent PIAS2 expression

• HepG2 cells: Human hepatocellular carcinoma cell line shows detectable PIAS2 expression

For neurological research applications, particularly those focused on Parkinson's disease models, neuronal cell lines with documented PIAS2 expression should be considered based on recent findings linking PIAS2 to neurodegenerative processes .

How should PIAS2 antibodies be stored to maintain reactivity?

Proper storage is critical for maintaining antibody performance. Based on manufacturer recommendations:

• Store at -20°C for most formulations; some require -80°C storage (particularly for conjugation-ready formats)

• Aliquot upon receipt to avoid repeated freeze-thaw cycles, which can significantly reduce antibody activity

• For antibodies in glycerol buffer (typically 50% glycerol), aliquoting is less critical for -20°C storage

• Short-term storage (1 month) at 2-8°C under sterile conditions after reconstitution is acceptable for most formats

• Long-term storage (6 months) requires -20°C to -70°C under sterile conditions after reconstitution

Note that conjugation-ready antibodies in PBS-only formulations (without BSA or sodium azide) have specific storage requirements and should be maintained at -80°C until immediately before conjugation .

How can I validate PIAS2 antibody specificity for critical experiments?

Rigorous validation is essential for experiments requiring high specificity. Implement multiple approaches:

The Human Protein Atlas enhanced validation approach, which involves comparing staining patterns using two independent antibodies with non-overlapping epitopes, provides particularly strong evidence for specificity .

What methodologies are recommended for studying PIAS2-protein interactions?

PIAS2 functions through protein-protein interactions in various cellular pathways. Several techniques are particularly useful:

• Proximity Ligation Assay (PLA): Detects protein-protein interactions in fixed cells with high sensitivity. PIAS2-CDKN2B interactions have been successfully visualized using this approach, with each red dot representing a protein-protein interaction complex in HeLa cells .

• Co-Immunoprecipitation (Co-IP): For studying PIAS2 interactions, use antibodies compatible with native protein conformation. The interaction between PIAS2 and UXT has been demonstrated using this approach by co-transfecting c-myc-tagged PIAS2 and GFP-tagged UXT into HEK 293T cells .

• Yeast Two-Hybrid Screening: Useful for identifying novel PIAS2 interaction partners. Studies have used PIAS2 (9-401 aa) as bait in pGBKT7 vector to identify interacting proteins .

• Fluorescence microscopy with tagged constructs: For visualizing subcellular co-localization, pDsRed-Express-1-PIAS2 and pEGFP-N1-tagged potential interacting proteins provide insights into spatial relationships .

When designing interaction studies, consider that PIAS2 interactions may be isoform-specific. For example, isoform PIAS2-beta promotes MDM2 sumoylation while isoform PIAS2-alpha does not .

How does PIAS2 upregulation affect neurodegenerative disease models?

Recent studies have established important connections between PIAS2 and neurodegenerative processes, particularly in Parkinson's disease:

• PIAS2 is upregulated in neurons of sporadic Parkinson's disease (sPD) patients, with particularly elevated levels in patients with dementia (sPDD)

• Overexpression of PIAS2 under a neuronal promoter in mice causes:

  • Motor and cognitive impairments

  • Intraneuronal phosphorylated α-synuclein accumulation

  • Dopaminergic neuron loss

  • Blocked mitophagy

  • Increased senescent mitochondria and oxidative stress

  • Inactivated ERK1/2-P53 signaling

When designing experiments to investigate PIAS2 in neurodegenerative contexts, consider:

• Using neuronal-specific promoters for targeted overexpression

• Implementing both in vitro and in vivo models to capture system-level effects

• Assessing both molecular markers (phosphorylated α-synuclein, oxidative stress indicators) and behavioral outcomes

• Exploring the relationship between PIAS2 and IFN-β signaling, as defective IFNβ-IFNAR1 signaling has been linked to PDD-like pathology

PIAS2 knockdown approaches may offer therapeutic insights, as they have shown rescue of clinicopathological manifestations of PDD in relevant models .

What are the critical considerations when detecting specific PIAS2 isoforms?

PIAS2 has multiple isoforms with distinct functions, requiring careful consideration of antibody selection and experimental design:

When studying isoform-specific functions:

• Use RT-PCR to confirm expression of specific isoform transcripts in your experimental system

• Consider using tagged constructs of specific isoforms for overexpression studies

• For Western blot analysis, use gradient gels (4-15%) to achieve better separation of closely migrating isoforms

• Validate observed bands with recombinant protein controls for each isoform

The choice of antibody is critical - ensure the epitope location allows discrimination between isoforms when this distinction is important to your research question .

How can I troubleshoot inconsistent PIAS2 antibody results across different applications?

When facing inconsistent results with PIAS2 antibodies across different applications, consider a systematic troubleshooting approach:

• Application-specific optimization:

  • Western blot: Test different lysis buffers as PIAS2 extraction efficiency varies; RIPA buffer with protease inhibitors is recommended

  • Immunofluorescence: Test different fixation methods, as some epitopes are fixation-sensitive; paraformaldehyde (4%) is generally effective

  • Immunohistochemistry: Optimize antigen retrieval methods; citrate buffer (pH 6.0) is often effective for PIAS2

• Sample-dependent factors:

  • Cell type variations: PIAS2 expression and localization differ between cell types; validate antibody performance in your specific cell type

  • Species reactivity: Confirm antibody cross-reactivity with your species of interest; most PIAS2 antibodies show human reactivity, with varying cross-reactivity to mouse, rat, and other species

  • Post-translational modifications: SUMOylation state may affect epitope accessibility; consider using phosphatase or SUMO-protease treatments to standardize samples

• Technical considerations:

  • Antibody concentration: Titrate antibody across a broader range than recommended (e.g., 1:100-1:5000) to determine optimal signal-to-noise ratio

  • Blocking reagents: Test alternative blocking agents (BSA, serum, commercial blockers) as background can vary significantly between applications

  • Detection systems: Compare different secondary antibodies and detection methods; HRP-conjugated secondary antibodies with ECL-Plus substrate work well for Western blot

Keep detailed records of all protocol variations to systematically identify critical parameters affecting PIAS2 detection in your specific experimental system.

What emerging applications are being developed for PIAS2 antibodies?

Recent advances in PIAS2 research are driving new applications for these antibodies:

• Multiplex imaging applications: Conjugation-ready PIAS2 antibody formats facilitate incorporation into multiplex imaging panels to study PIAS2 alongside other proteins in complex tissues

• Cytometric bead arrays: Matched antibody pairs enable development of quantitative assays for PIAS2 across multiple samples, particularly valuable for clinical research

• Mass cytometry (CyTOF): Metal-conjugated PIAS2 antibodies allow integration into high-dimensional single-cell protein profiling panels

• Therapeutic target validation: Given PIAS2's role in neurodegenerative processes, antibodies are increasingly used to validate it as a potential therapeutic target

As research continues to uncover PIAS2's roles in disease processes, particularly in neurodegeneration, the applications for these antibodies will likely expand into diagnostics and therapeutic development pathways.

How should researchers evaluate contradictory findings when using different PIAS2 antibodies?

When faced with contradictory results using different PIAS2 antibodies:

• Compare antibody characteristics:

  • Epitope location: Different domains may have different accessibility or be isoform-specific

  • Clonality: Monoclonal antibodies target single epitopes while polyclonals recognize multiple epitopes

  • Host species: May affect background in certain applications

  • Production method: Recombinant antibodies offer better batch-to-batch consistency

• Implement orthogonal validation methods:

  • Genetic approaches (siRNA, CRISPR)

  • mRNA expression analysis

  • Mass spectrometry validation

  • Independent technique confirmation

• Consider biological variables:

  • PIAS2 undergoes dynamic SUMOylation and other post-translational modifications

  • Nuclear-cytoplasmic shuttling affects detection in subcellular fractions

  • Expression levels vary significantly across tissues and cell states