puf60b Antibody

What is PUF60 and what biological functions does it serve?

PUF60 (poly-U binding splicing factor 60KDa) is a multifunctional protein involved in several critical cellular processes. It primarily functions in RNA splicing, particularly under suboptimal conditions, and serves as a polyU-binding factor required for optimal RNA processing . Beyond splicing, PUF60 participates in transcriptional regulation and may modulate the location or function of non-coding RNAs such as hYRNA . The protein exists in multiple isoforms with theoretical molecular weights of approximately 59.9, 58.2, and 55.7 kDa . PUF60 has gained significant research interest due to its interactions with other nuclear proteins, many of which are known autoantigens, and its potential role in cancer biology, as multiple cancers show amplification of the PUF60 locus and elevated expression of the protein .

What are the primary applications for PUF60 antibodies in research?

PUF60 antibodies serve multiple experimental purposes in research settings:

• Western Blot (WB): Recommended dilutions range from 1:500 to 1:3000, allowing detection of endogenous PUF60 protein in cell and tissue lysates

• Immunohistochemistry (IHC): Used at dilutions of 1:50 to 1:200 for tissue section analysis

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Applied at dilutions of 1:50 to 1:200 for subcellular localization studies

• Enzyme-linked Immunosorbent Assay (ELISA): High sensitivity applications at dilutions up to 1:32000

• Immunoprecipitation: Effective for pulling down PUF60 and associated protein complexes for interaction studies

The optimal working dilution should be determined by the researcher based on specific experimental conditions and sample types .

How should PUF60 antibodies be stored and handled to maintain reactivity?

For optimal maintenance of antibody activity, PUF60 antibodies should be:

• Stored at -20°C for long-term preservation

• Aliquoted upon receipt to avoid repeated freeze-thaw cycles that can compromise antibody integrity

• Handled with caution if the preparation contains sodium azide, which is a hazardous substance requiring trained personnel for safe handling

• Centrifuged briefly if small volumes become entrapped in the vial seal during shipment

• Maintained in their appropriate storage buffer (typically Tris saline, pH 7.3 with 0.5% BSA and 0.02% sodium azide, or similar stabilizing solutions containing glycerol)

Proper storage and handling significantly extend the functional lifespan of these reagents and ensure consistent experimental results.

What controls should be included when using PUF60 antibodies in experimental protocols?

A robust experimental design with PUF60 antibodies should incorporate several critical controls:

These controls are essential for validating experimental results and distinguishing genuine PUF60 signals from artifacts or non-specific background . For autoimmune studies involving PUF60 as an autoantigen, including healthy control sera is particularly important for establishing baseline reactivity .

How can researchers optimize Western blot protocols for detecting PUF60 variants?

Optimizing Western blot protocols for PUF60 detection requires attention to several technical considerations:

• Sample Preparation: Use RIPA buffer for effective protein extraction from tissues and cell lines . For complex samples, consider subcellular fractionation to enrich nuclear proteins.

• Protein Loading: Load approximately 35μg of total protein per lane for cell lysates, adjusting as needed based on PUF60 expression levels in your experimental system .

• Gel Selection: Use 10-12% polyacrylamide gels to achieve optimal separation of PUF60 isoforms (theoretical MWs: 59.9, 58.2, and 55.7 kDa) .

• Transfer Conditions: Implement semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C for more efficient transfer of higher molecular weight proteins.

• Blocking Strategy: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Antibody Incubation: Dilute primary antibody (1-3 μg/mL) and incubate for 1 hour at room temperature or overnight at 4°C .

• Detection Method: Chemiluminescence provides sensitive detection , with enhanced sensitivity achieved through signal amplification systems for low-abundance proteins.

For optimal separation of closely related isoforms, consider using 2D gel electrophoresis followed by Western blotting, as demonstrated in the identification of PUF60 as an autoantigen .

What are the methodological considerations for detecting PUF60 autoantibodies in patient samples?

Detection of anti-PUF60 autoantibodies in patient sera requires specialized approaches:

• ELISA Protocol Development:

  • Coat plates with recombinant human PUF60 protein at 1-2 μg/mL

  • Use diluted patient sera (typically 1:100 to 1:1000)

  • Incorporate multiple washing steps to minimize background

  • Include known positive and negative control sera for standardization

• Immunoprecipitation-Western Blot (IP-WB):

  • Immunoprecipitate PUF60 from cell lysates using patient sera

  • Follow with Western blotting using commercial anti-PUF60 antibodies

  • This approach confirms that autoantibodies specifically target PUF60

• Distinguishing Cross-Reactivity:

  • Immunodepletion experiments should be performed when patients have multiple autoantibodies

  • This helps determine whether apparent co-reactivity is due to antibody cross-reactivity or truly distinct antibody populations

• Sample Cohort Considerations:

  • Include diverse disease groups and healthy controls

  • In published studies, anti-PUF60 antibodies were found in 30% of Sjögren's syndrome patients, 18% of dermatomyositis patients, and approximately 5% of healthy controls

These methodological approaches have been successfully employed to establish PUF60 as a significant autoantigen in autoimmune diseases, particularly in Sjögren's syndrome and dermatomyositis .

How does PUF60 interact with other RNA-binding proteins and what methods best capture these interactions?

PUF60 participates in complex protein-protein interaction networks, particularly with other RNA-binding proteins and splicing factors. Several methodologies can effectively capture these interactions:

• Co-immunoprecipitation (Co-IP): PUF60 antibodies can pull down associated proteins like Ro60, with which it has demonstrated biochemical and biological interactions . The complex can then be analyzed by Western blotting or mass spectrometry.

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ, offering spatial resolution of where PUF60 interactions occur within cellular compartments.

• Crosslinking and Immunoprecipitation (CLIP): For studying RNA-protein interactions, CLIP methods reveal the RNA targets of PUF60 in vivo.

• Yeast Two-Hybrid Screening: Can identify novel protein partners of PUF60, though results should be confirmed with co-IP or other methods.

• Bimolecular Fluorescence Complementation (BiFC): Allows visualization of protein interactions in living cells through the reconstitution of a fluorescent protein.

Research has shown that PUF60 interacts with several known autoantigens, including Ro60, as well as components of the U2 snRNP complex involved in RNA splicing . These interactions may explain the targeting of these proteins in autoimmune diseases through epitope spreading mechanisms.

What is the significance of PUF60 as an autoantigen in different autoimmune diseases?

The emergence of PUF60 as an autoantigen presents intriguing disease-specific patterns:

The disease-specific associations are particularly noteworthy. In Sjögren's syndrome, anti-PUF60 antibodies cluster with traditional SS-associated autoantibodies (anti-Ro/La), while in dermatomyositis, they associate with anti-TIF-1γ antibodies but not other DM-specific antibodies . This suggests that different immunogenic mechanisms may drive anti-PUF60 responses in different disease contexts, possibly related to tissue-specific expression patterns or protein-protein interactions unique to each disease microenvironment .

How can researchers address specificity issues when multiple isoforms of PUF60 exist?

Addressing specificity challenges with multiple PUF60 isoforms requires strategic experimental approaches:

• Isoform-Specific Antibody Selection: Some commercial antibodies, such as the Abnova PAB6268, are designed to recognize multiple human isoforms (NP_055096.2; NP_510965.1) . When more selective detection is needed, researchers should:

  • Review the immunogen sequence to determine which isoforms will be recognized

  • Consider custom antibody production against unique peptide sequences when isolating specific isoforms

• Electrophoretic Separation:

  • Standard SDS-PAGE can separate PUF60 isoforms (59.9, 58.2, and 55.7 kDa)

  • Higher resolution separation requires 2D gel electrophoresis, which was successfully employed in PUF60 autoantigen identification

• Recombinant Protein Controls:

  • Express individual recombinant isoforms as positive controls

  • Use these standards to validate isoform-specific detection

• RNA Expression Analysis:

  • Complement protein studies with RT-PCR or RNA-Seq to identify which isoform transcripts are expressed

  • Design primers spanning unique exon junctions for isoform-specific amplification

• Mass Spectrometry:

  • Identify isoform-specific peptides through proteomics approaches

  • This technique definitively identified PUF60 in autoimmune studies

What are common technical challenges when using PUF60 antibodies and how can they be resolved?

Researchers frequently encounter several technical issues when working with PUF60 antibodies:

• High Background in Immunohistochemistry/Immunofluorescence:

  • Problem: Non-specific staining obscuring specific PUF60 signal

  • Solution: Increase blocking time/concentration (5% BSA or normal serum), optimize antibody dilution (start at 1:50 and titrate) , and include additional washing steps with 0.1% Tween-20

• Multiple Bands in Western Blots:

  • Problem: Distinguishing between specific isoforms, degradation products, and non-specific binding

  • Solution: Include recombinant PUF60 as a positive control, use freshly prepared lysates with protease inhibitors, and perform peptide competition assays to identify specific bands

• Weak Signal Detection:

  • Problem: Insufficient sensitivity for low-abundance PUF60

  • Solution: Increase protein loading (up to 50μg), extend primary antibody incubation time (overnight at 4°C), and use signal enhancement systems such as biotin-streptavidin amplification

• Cross-Reactivity Issues:

  • Problem: Antibody recognizing non-target proteins

  • Solution: Validate antibody specificity using PUF60 knockout/knockdown controls, and confirm results with multiple antibodies targeting different epitopes

• Inconsistent Immunoprecipitation Results:

  • Problem: Variable efficiency in pulling down PUF60 complexes

  • Solution: Optimize lysate preparation (adjust salt concentration), pre-clear lysates thoroughly, and consider crosslinking to stabilize transient interactions

These technical challenges can be systematically addressed through careful optimization of experimental conditions and appropriate controls.

How should researchers interpret contradictory results between different detection methods for PUF60?

When faced with conflicting results across different detection methods:

• Methodological Sensitivity Analysis:

  • Different techniques have varying detection thresholds

  • Western blotting may detect denatured epitopes missed by immunofluorescence techniques that preserve native conformation

  • ELISA might detect lower abundance proteins than Western blotting

• Epitope Accessibility Considerations:

  • Discrepancies often result from differential epitope exposure

  • Fixation methods in IHC/IF may mask epitopes recognized by certain antibodies

  • Solution: Try multiple fixation methods or antigen retrieval techniques

• Isoform-Specific Detection:

  • Different methods may preferentially detect certain PUF60 isoforms

  • Review antibody documentation for isoform specificity (e.g., the Abnova antibody recognizes both reported human isoforms)

  • Consider using isoform-specific primers in RT-PCR to complement protein studies

• Cellular Localization Effects:

  • Nuclear proteins like PUF60 may require specific extraction methods

  • Contradictory results might reflect differences in extraction efficiency rather than actual protein levels

  • Solution: Use subcellular fractionation to confirm localization

• Reconciliation Approaches:

  • When possible, utilize orthogonal methods (e.g., mass spectrometry) to resolve contradictions

  • Apply quantitative approaches like densitometry (Western blots) or fluorescence intensity measurements (IF) for objective comparisons

  • Consider cell/tissue-specific expression patterns that might explain method-dependent variations

How can researchers differentiate between autoantibodies targeting PUF60 and those targeting related proteins in clinical samples?

Distinguishing between autoantibodies to PUF60 and related proteins is methodologically challenging but critical for accurate interpretation:

• Sequential Immunodepletion Studies:

  • Sequentially deplete sera of specific autoantibodies and re-test for remaining reactivities

  • Research has shown that when PUF60 antibodies are immunodepleted from sera also containing antibodies against TIF-1γ, Ro52, or Ro60, the other reactivities remained unchanged, confirming these are distinct antibody populations rather than cross-reactive antibodies

• Competitive ELISA Approaches:

  • Pre-incubate sera with soluble recombinant proteins (PUF60, Ro60, etc.)

  • Test for inhibition of binding to plate-bound antigens

  • Specific inhibition indicates antibody specificity, while cross-inhibition suggests cross-reactivity

• Epitope Mapping:

  • Use peptide arrays or truncated protein constructs to identify specific epitopes recognized by patient antibodies

  • This can distinguish between antibodies targeting shared domains versus unique regions

• Absorption Studies:

  • Absorb sera against purified recombinant proteins immobilized on solid supports

  • Test depleted sera for remaining reactivities against different antigens

• Recombinant Protein Panel Testing:

  • Test sera against a panel of purified recombinant proteins in parallel

  • Analyze reactivity patterns to identify specific versus cross-reactive responses

These approaches were successfully employed in research establishing PUF60 as a distinct autoantigen in Sjögren's syndrome and dermatomyositis, demonstrating that co-occurrence of anti-PUF60 with other autoantibodies represents distinct immune responses rather than cross-reactivity .

What emerging applications of PUF60 antibodies might advance our understanding of RNA processing disorders?

Several innovative applications of PUF60 antibodies could drive forward our understanding of RNA processing pathologies:

• Single-Cell Immunoprofiling:

  • Combining PUF60 antibodies with single-cell technologies could reveal cell-specific expression patterns

  • This may identify vulnerable cell populations in splicing-related disorders

  • Integration with transcriptomics could correlate PUF60 expression with alternative splicing events

• Spatial Transcriptomics Integration:

  • Coupling PUF60 immunodetection with spatial transcriptomics

  • This could map relationships between PUF60 localization and tissue-specific splicing patterns

  • Particularly relevant for understanding region-specific RNA processing in neurological disorders

• Dynamic Interaction Mapping:

  • Using PUF60 antibodies in live-cell imaging with other fluorescently tagged splicing factors

  • This approach could reveal the kinetics of spliceosome assembly and function

  • Potential applications in studying how splicing dynamics are altered in disease states

• Therapeutic Monitoring:

  • PUF60 antibodies as biomarkers for monitoring response to treatments targeting RNA processing

  • Quantitative analysis of PUF60 expression or localization changes following therapy

  • Potential application in personalized medicine approaches for splicing-related disorders

• Multi-omics Integration:

  • Combining PUF60 antibody-based proteomics with RNA-seq and CLIP-seq

  • This integrated approach could provide comprehensive views of how PUF60 coordinates RNA processing

  • Especially valuable for complex diseases with RNA processing aberrations

These emerging applications represent frontier opportunities for expanding our understanding of the role of PUF60 in normal physiology and disease pathogenesis.

How might the study of PUF60 autoantibodies contribute to personalized medicine approaches for autoimmune diseases?

The discovery of PUF60 as an autoantigen opens several avenues for personalized medicine in autoimmune disease management:

• Patient Stratification:

  • Anti-PUF60 antibodies show disease-specific patterns of association with other autoantibodies (Ro52/Ro60/La in Sjögren's syndrome; TIF-1γ in dermatomyositis)

  • These patterns could define patient subgroups with distinct prognoses or treatment responses

  • Research shows that in dermatomyositis, anti-PUF60 antibodies are associated with Caucasian race, suggesting potential genetic/ethnic factors in autoimmune responses

• Predictive Biomarkers:

  • Longitudinal studies tracking anti-PUF60 antibody levels before and during disease progression

  • Could identify whether these antibodies predict disease onset, flares, or complications

  • Particularly relevant given the association with hyperglobulinemia in Sjögren's syndrome

• Treatment Selection Biomarkers:

  • Correlating anti-PUF60 antibody status with treatment outcomes

  • Could guide selection of immunomodulatory therapies

  • Potential for monitoring changes in antibody levels as markers of treatment efficacy

• Novel Therapeutic Targets:

  • Understanding PUF60's role in autoimmunity might reveal targetable immunological pathways

  • Epitope-specific approaches could selectively modulate pathogenic immune responses

  • Potential for tolerance induction therapies specific to PUF60 epitopes

• Early Diagnosis:

  • With 30% prevalence in Sjögren's syndrome and 18% in dermatomyositis, anti-PUF60 antibodies might enable earlier diagnosis when added to diagnostic panels

  • This could facilitate early intervention before irreversible tissue damage occurs

The disease-specific associations of anti-PUF60 antibodies suggest they may represent distinct pathogenic mechanisms in different autoimmune conditions, potentially informing tailored therapeutic approaches for patient subgroups .