Snrnp35 Antibody

What is SNRNP35 and what is its functional role in cellular processes?

SNRNP35 (Small Nuclear Ribonucleoprotein 35) is a critical component of the U11/U12 small nuclear ribonucleoproteins (snRNP) that constitute part of the U12-type spliceosome. It functions as a homolog of the U1-snRNP binding protein. The protein has a distinctive structure with an N-terminal half containing an RNA recognition motif (RRM) and a C-terminal half rich in Arg/Asp and Arg/Glu dipeptide repeats, which is characteristic of various splicing factors . These structural features enable SNRNP35 to participate in pre-mRNA processing through the minor spliceosome pathway.

The gene encoding SNRNP35 undergoes alternative splicing, resulting in multiple transcript variants that produce two distinct protein isoforms along with non-protein coding variants . As part of the minor spliceosome complex, SNRNP35 is essential for the splicing of a subset of introns with non-canonical splice sites, contributing to RNA processing diversity in eukaryotic cells.

What are the standard applications for SNRNP35 antibodies in molecular biology research?

SNRNP35 antibodies are versatile reagents employed in several key molecular biology techniques:

• Western Blotting (WB): The primary application for detecting SNRNP35 protein expression levels and examining post-translational modifications. Most commercial antibodies are validated for WB applications with typical dilution ranges of 1:500-1:2000 .

• Immunohistochemistry (IHC): Used for visualizing SNRNP35 expression and localization in tissue sections, providing insights into tissue-specific expression patterns .

• Immunocytochemistry (ICC): Applied for cellular localization studies, confirming the predominantly nuclear localization of SNRNP35 .

• ELISA (Enzyme-Linked Immunosorbent Assay): Utilized for quantitative detection of SNRNP35 in various sample types .

• Immunoprecipitation: Employed for isolating SNRNP35 and its interaction partners from complex protein mixtures, particularly valuable in studying spliceosome complex assembly .

What is the expected molecular weight of SNRNP35 in Western blot analysis and how should researchers interpret band variations?

While the calculated molecular weight of SNRNP35 is approximately 29 kDa, the protein typically appears as a 35 kDa band in Western blot analysis . This discrepancy between calculated and observed molecular weights is not uncommon in protein analysis and can be attributed to several factors:

• Post-translational modifications affecting protein mobility

• Structural properties influencing migration patterns

• Alternative splicing generating different isoforms

As noted in product documentation: "The actual band is not consistent with the expectation. Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

Researchers should be aware that if a protein sample contains different modified forms simultaneously, multiple bands may be detected on the membrane. Validation with positive controls and examination of additional markers can help confirm specificity.

How do SNRNP35 antibodies contribute to understanding autoimmune conditions like scleroderma?

Research has revealed an important connection between SNRNP35 and autoimmune conditions, particularly scleroderma. A systematic autoantigen analysis identified that approximately 25% of scleroderma patients without known autoantibody specificities had autoantibodies targeting components of the minor spliceosome complex, including RNPC3, PDCD7, SNRNP25, and SNRNP35 .

In these investigations, researchers discovered that serum from these patients either recognized all four specificities (RNPC3, PDCD7, SNRNP25, and SNRNP35) or were negative for all, suggesting coordinated autoimmune responses against the entire minor spliceosome complex . This finding indicates epitope spreading of the autoantibody response within the spliceosome complex, similar to what has been observed with POLR3 complex autoantibodies in other scleroderma subtypes.

Methodologically, researchers can use SNRNP35 antibodies for:

• Identifying patient subgroups with specific autoantibody profiles

• Studying epitope spreading mechanisms in autoimmune conditions

• Developing biomarkers for disease subsets in precision medicine approaches

What methodological considerations should be taken when using different detection platforms with SNRNP35 antibodies?

Different detection platforms show varying sensitivity and specificity for SNRNP35 antibodies, which has significant methodological implications:

PhIP-Seq vs. PLATO vs. Immunoprecipitation Comparison:

Research has shown that while PhIP-Seq (Phage Immunoprecipitation Sequencing) successfully detected some components of the minor spliceosome complex like RNPC3 and PDCD7, it failed to detect SNRNP25 and SNRNP35 in patient samples . In contrast, PLATO (Parallel Analysis of Translated ORFs) identified all four components, including SNRNP25 and SNRNP35 .

The difference in detection sensitivity was attributed to:

• PLATO's enhanced sensitivity for detecting antibodies against discontinuous epitopes

• The relatively smaller size of SNRNP25 and SNRNP35 proteins, presenting fewer opportunities for linear epitopes that PhIP-Seq detects

These findings suggest that PLATO may be more effective for proteins where conformational epitopes are important for antibody recognition. As noted in the research: "Interestingly, SNRNP25 and SNRNP35 were not detected in the PhIP-Seq screens, perhaps because PLATO is more sensitive for detecting antibodies against discontinuous epitopes and these two proteins are shorter, with fewer opportunities for linear epitopes" .

Traditional immunoprecipitation with IVTT (In Vitro Transcription and Translation)-generated proteins confirmed results from PLATO, with 87.5% agreement for some components but lower agreement for others, indicating technological complementarity rather than redundancy.

What approaches can researchers use to validate the specificity of SNRNP35 antibodies in experimental systems?

Ensuring antibody specificity is critical for reliable experimental outcomes. For SNRNP35 antibodies, researchers should consider these validation approaches:

• Multiple Epitope Targeting: Commercial SNRNP35 antibodies target different regions of the protein, including:

  • AA 1-150 (N-terminal region containing the RNA recognition motif)

  • AA 1-244 (full-length protein)

  • AA 29-78 (internal epitope)

  • N-terminal region

• Cross-Reactivity Assessment: Most anti-SNRNP35 antibodies show cross-reactivity with human and mouse proteins, with some also reacting with additional species like rabbit, cow, dog, guinea pig, horse, and rat . Researchers should verify cross-reactivity for their specific experimental model.

• Statistical Validation: In larger studies, statistical methods like false discovery rate analysis based on permutation analysis of Fisher's exact P values can help validate enrichment of SNRNP35 antibodies in specific patient cohorts .

How can machine learning approaches enhance antibody-antigen prediction for SNRNP35 research?

Recent advances in machine learning have opened new avenues for improving antibody-antigen binding prediction, with potential applications for SNRNP35 research:

Active learning methodologies have shown promise in enhancing out-of-distribution lab-in-the-loop prediction tasks. These approaches are particularly valuable when:

• Dealing with library-on-library approaches: Where many antigens (including SNRNP35) are probed against many antibodies to identify specific interacting pairs .

• Addressing out-of-distribution prediction challenges: When models need to predict interactions involving antibodies and antigens not represented in training data .

• Optimizing experimental efficiency: Recent research demonstrated that active learning algorithms could reduce the number of required antigen mutant variants by up to 35% and accelerate the learning process compared to random baseline approaches .

The methodological implementation involves:

• Starting with a small labeled subset of antibody-antigen binding data

• Iteratively expanding the labeled dataset based on model uncertainty

• Evaluating multiple active learning strategies (fourteen were compared in one study)

• Using simulation frameworks like Absolut! to evaluate performance prior to wet-lab validation

Such computational approaches could significantly reduce the experimental cost and time required for developing and characterizing SNRNP35 antibodies with optimal binding properties.

What are the recommended storage and handling protocols for maintaining SNRNP35 antibody activity?

Optimal storage and handling of SNRNP35 antibodies is essential for maintaining their activity and specificity:

Storage Conditions:

• Store at -20°C for long-term storage (valid for 12 months)

• Avoid repeated freeze/thaw cycles which can degrade antibody activity

• Some formulations may be stored at +4°C for short-term use

Buffer Composition:
Most commercial SNRNP35 antibodies are supplied in:

• Phosphate buffered solution, pH 7.4

• Containing 0.05% stabilizer

• With 50% glycerol to prevent freeze damage

Shipping Considerations:

• Typically shipped with ice packs

• Upon receipt, store immediately at the recommended temperature

Working Dilutions:

• For Western blotting: 1:500-1:2000 dilution is generally recommended

• Optimal dilutions should be determined empirically for each specific application

How can researchers optimize immunoprecipitation protocols for studying SNRNP35's interaction with other spliceosome components?

Optimizing immunoprecipitation (IP) protocols is crucial when studying SNRNP35's interactions within the spliceosome complex:

• Protein Expression System Selection:
  Research has demonstrated success using IVTT (In Vitro Transcription and Translation) systems to generate SNRNP35 and other spliceosome components for immunoprecipitation studies . This approach is particularly useful when studying autoantibody recognition of multiple spliceosome components.

What are the critical quality control parameters for evaluating SNRNP35 antibodies before experimental use?

Thorough quality control is essential before using SNRNP35 antibodies in critical experiments:

• Specificity Verification:

  • Validate the antibody using positive controls expressing SNRNP35

  • Confirm the expected molecular weight (typically appears at 35 kDa despite calculated MW of 29 kDa)

  • Consider knockout/knockdown controls to confirm specificity

• Cross-Reactivity Assessment:

  • Verify species cross-reactivity relevant to your experimental system

  • Most commercial antibodies react with human and mouse SNRNP35, with varying reactivity to other species

  • Test across relevant cell lines (most antibodies are verified across "various cell lines")

• Application-Specific Validation:

  • Confirm performance in your specific application (WB, IHC, ICC, ELISA)

  • Optimize conditions including dilution, incubation time, and detection method

  • For nuclear proteins like SNRNP35, ensure proper nuclear extraction and sample preparation

• Batch-to-Batch Consistency:

  • Request certificate of analysis for lot-specific performance data

  • Consider testing multiple lots in parallel for critical experiments

  • Maintain reference samples for longitudinal quality control

By implementing these quality control measures, researchers can ensure reliable and reproducible results when working with SNRNP35 antibodies across various experimental contexts.

What role can SNRNP35 antibodies play in developing new biomarkers for autoimmune disease subtypes?

SNRNP35 antibodies have demonstrated significant potential as biomarkers for specific autoimmune disease subtypes:

Research has identified a distinct subgroup of scleroderma patients (approximately 25%) who have autoantibodies against RNPC3 and other minor spliceosome components including SNRNP35 . This finding is particularly significant because:

• It identifies a previously uncharacterized patient subgroup: "We found that 25% of these patients had autoantibodies to RNA Binding Region Containing 3 (RNPC3) and multiple other components of the minor spliceosome."

• It demonstrates specificity to a particular autoimmune condition: The autoantibodies against the minor spliceosome complex "is found in patients with scleroderma without known specificities and is absent in unrelated autoimmune diseases."

The research methodologies employed include:

• High-throughput antibody epitope discovery methods

• Comparative analysis against control samples (123 control samples, including samples from healthy donors and patients with other autoimmune diseases)

• Statistical validation using false discovery rate calculations

This approach exemplifies how SNRNP35 antibodies can contribute to the development of precision medicine biomarkers that help stratify patients into more homogeneous subgroups, potentially leading to more targeted therapeutic approaches.