SYNCRIP Antibody

What is SYNCRIP and what are its alternative nomenclatures?

SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that primarily localizes in the cytoplasm. It is also identified as hnRNP Q, NSAP1, PP68, GRY-RBP, GRYRBP, HNRNPQ, and HNRPQ1 . The protein has a calculated molecular weight of approximately 70 kDa, though it is typically observed between 62-74 kDa in experimental conditions . Structurally, SYNCRIP contains an N-terminal acidic domain, three RNA-binding domains, an RGG box (another type of RNA-binding motif), and a C-terminal tyrosine-rich motif that mediates protein-protein interactions .

What are the primary biological functions of SYNCRIP?

SYNCRIP is involved in multiple aspects of RNA metabolism and cellular processes:

• mRNA processing mechanisms, including splicing and translation regulation

• Component of the CRD-mediated complex that promotes MYC mRNA stability

• Part of the APOB mRNA editosome complex that may modulate postranscriptional C to U RNA-editing of APOB mRNA through binding to A1CF (APOBEC1 complementation factor), APOBEC1, or RNA itself

• Involvement in translationally coupled mRNA turnover

• Participation in cytoplasmic deadenylation/translational and decay interplay of FOS mRNA mediated by the major coding-region determinant of instability (mCRD) domain

• Regulation of viral RNA replication for viruses such as mouse hepatitis virus (MHV) and hepatitis C virus (HCV)

• Promotion of proliferation in colorectal cancer cells

Which experimental applications are most appropriate for SYNCRIP antibodies?

SYNCRIP antibodies have been validated for multiple applications, with varying dilution recommendations based on the specific application:

It is essential to optimize these dilutions for each specific experimental system to achieve optimal results . Researchers should consider that antibody performance may vary across different cell types and tissues.

How should I optimize SYNCRIP antibody usage for Western blot applications?

For optimal Western blot results with SYNCRIP antibodies:

• Start with a dilution range of 1:500-1:2000 for initial optimization .

• Be aware that SYNCRIP typically appears between 62-74 kDa on SDS-PAGE gels, though the calculated molecular weight is 70 kDa .

• The upper protein bands of approximately 85 and 74 kDa may represent hnRNP R and its degradation product, which can weakly cross-react with anti-SYNCRIP antibody .

• For sample preparation, consider that SYNCRIP is associated with detergent-resistant membrane fractions (DRM), which may affect extraction efficiency .

• When analyzing SYNCRIP knockdown experiments, account for the stability of SYNCRIP protein—a lag time of more than 1 day has been observed between SYNCRIP reduction and subsequent effects on target RNAs such as HCV RNA .

How can SYNCRIP antibodies be used to study RNA-protein interactions?

To investigate SYNCRIP-RNA interactions using antibodies:

• UV Cross-linking and Immunoprecipitation: This approach has been successfully used to demonstrate SYNCRIP binding to both positive- and negative-strand UTRs of MHV RNA. The procedure involves:

  • UV cross-linking cell extracts with 32P-labeled RNA

  • Immunoprecipitation with anti-SYNCRIP antibody

  • Analysis of the cross-linked complexes by SDS-PAGE

• RNA Affinity Purification: SYNCRIP was initially identified using biotin-labeled 5'-UTR RNA affinity purification followed by mass spectrometry (MALDI-MS). This method can be used to verify RNA-binding specificity of SYNCRIP to different RNA targets .

• Mapping RNA-binding Sites: After establishing binding, researchers can map the specific binding sites using deletion mutants of the RNA. For example, SYNCRIP binding to MHV RNA was mapped to the leader sequence of the 5'-UTR, requiring the UCUAA repeat sequence .

What approaches can be used to study SYNCRIP's role in viral replication mechanisms?

To investigate SYNCRIP's function in viral replication:

• siRNA Knockdown followed by Viral RNA Quantification:

  • Transfect cells with SYNCRIP-specific siRNA

  • Infect with virus or transfect with viral replicon

  • Quantify viral RNA by qRT-PCR at various time points

  • Monitor SYNCRIP protein levels by Western blot

• In vitro Replication Assay with Immunodepletion:

  • Isolate detergent-resistant membrane fractions from cells containing viral replicons

  • Immunodeplete SYNCRIP using specific antibodies

  • Perform in vitro replication assay to directly assess SYNCRIP's role in viral RNA synthesis

  • Include appropriate controls (non-specific IgG, other relevant antibodies)

• Colocalization Studies:

  • Transfect cells with fluorescently tagged SYNCRIP (e.g., peGFP-SYNCRIP)

  • Label newly synthesized viral RNA using BrUTP in actinomycin D-pretreated cells

  • Perform immunofluorescence with anti-BrdU antibody

  • Analyze colocalization patterns by confocal microscopy

How can I distinguish SYNCRIP's effects on RNA replication from its effects on translation?

This is a critical methodological challenge when studying multifunctional proteins like SYNCRIP. Researchers have addressed this using:

• In vitro Replication Assay: This approach separates replication from translation by using crude membrane fractions after SYNCRIP knockdown or immunodepletion. The assay directly measures RNA synthesis without the confounding effects of translation .

• Translation-specific Assays:

  • In vitro: Incubate rabbit reticulocyte lysate with radiolabeled and in vitro-transcribed RNA (such as MHV defective interfering RNA) with increasing amounts of purified SYNCRIP protein. Analyze 35S-labeled translation products by SDS-PAGE .

  • In vivo: Transfect cells with reporter constructs containing viral UTRs (e.g., MHV-UTR/LUC) in SYNCRIP-overexpressing or SYNCRIP-knockdown cells, followed by luciferase assay .

• Temporal Analysis: Monitor the timing of effects after SYNCRIP knockdown. If translation is primarily affected, viral protein synthesis would decrease before viral RNA levels. In contrast, direct effects on replication would show concurrent changes in RNA levels .

How is SYNCRIP involved in colorectal cancer progression, and how can antibodies help investigate this role?

Recent research has identified SYNCRIP as highly expressed in colorectal cancer (CRC), with significant implications for tumor growth:

• Expression Analysis: TCGA and GEPIA database analysis confirmed SYNCRIP overexpression in CRC tumors compared to normal tissues. This finding was validated using anti-SYNCRIP antibodies in Western blot and immunohistochemistry of CRC tissue samples .

• Functional Role:

  • SYNCRIP depletion using shRNA in CRC cell lines (SW480 and HCT 116) resulted in increased caspase3/7 activity and decreased cell proliferation and migration

  • Overexpression of SYNCRIP showed opposite effects, promoting proliferation

  • In vivo experiments demonstrated that SYNCRIP depletion significantly inhibited colorectal tumor growth

• Mechanistic Insights: SYNCRIP regulated the expression of DNA methyltransferase (DNMT) 3A, but not DNMT1 or DNMT3B, which affected the expression of tumor suppressor p16 .

To study SYNCRIP in cancer research:

• Use anti-SYNCRIP antibodies for immunohistochemistry of patient tumor samples (recommended dilution 1:1000-1:4000)

• Perform Western blot analysis of cancer cell lines to compare SYNCRIP expression levels

• Employ immunofluorescence to examine subcellular localization in cancer vs. normal cells

• Combine with functional studies (knockdown, overexpression) to correlate expression with phenotypic effects

What are the technical considerations when using SYNCRIP antibodies in cancer tissue immunohistochemistry?

For optimal SYNCRIP immunohistochemistry in cancer tissues:

• Antigen Retrieval: Use TE buffer pH 9.0 for optimal results, though citrate buffer pH 6.0 may serve as an alternative .

• Antibody Dilution: Begin with a dilution range of 1:1000-1:4000, and optimize based on your specific tissue samples and detection system .

• Positive Controls: Human colon cancer tissue has been validated as a positive control for SYNCRIP immunostaining .

• Interpretation Challenges: Be aware that SYNCRIP expression may vary across different cancer types and even within the same tumor. Consider using multiple antibody clones targeting different epitopes of SYNCRIP to validate findings and minimize potential artifacts.

• Correlation with Clinical Data: When analyzing SYNCRIP expression in patient samples, carefully correlate with clinical parameters to establish potential prognostic value.

How should I address cross-reactivity concerns when using SYNCRIP antibodies?

SYNCRIP antibodies may cross-react with related proteins, particularly other hnRNP family members:

• Known Cross-reactivity: Anti-SYNCRIP antibodies may cross-react with hnRNP R and its degradation products, appearing as bands of approximately 85 and 74 kDa in Western blots .

• Validation Approaches:

  • Use siRNA or shRNA knockdown of SYNCRIP to confirm specificity of antibody binding

  • Include recombinant SYNCRIP protein as a positive control

  • Compare reactivity patterns across multiple anti-SYNCRIP antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

• Species Considerations: When working across species, note that while human and mouse SYNCRIP are highly conserved, there may be differences in antibody reactivity. Validate each antibody for cross-species applications .

What are the critical factors for reproducible immunoprecipitation of SYNCRIP-RNA complexes?

For robust immunoprecipitation of SYNCRIP-RNA complexes:

• Antibody Selection: Use antibodies specifically validated for immunoprecipitation. For example, some anti-SYNCRIP antibodies are recommended at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate .

• Cross-linking Optimization:

  • For protein-protein interactions, chemical cross-linkers like formaldehyde or DSP can be used

  • For RNA-protein interactions, UV cross-linking (254 nm) provides specificity but lower efficiency

  • The duration and intensity of UV exposure should be optimized for each experimental system

• Buffer Conditions:

  • SYNCRIP is associated with detergent-resistant membrane fractions, which may require specialized extraction conditions

  • Consider using TX-100 at 4°C for 30 min followed by sucrose gradient centrifugation to isolate detergent-resistant membrane fractions containing SYNCRIP

• Controls:

  • Include non-specific IgG as a negative control

  • If possible, use SYNCRIP-depleted extracts as an additional control

  • Consider including RNase treatment to confirm RNA-dependency of interactions

How might emerging technologies enhance SYNCRIP antibody applications in research?

Several emerging technologies could significantly advance SYNCRIP research:

• Proximity Labeling Approaches: BioID or APEX2 fusions with SYNCRIP could identify novel protein interaction partners in living cells, providing insights into its functional complexes without reliance on antibody-based pull-downs.

• Single-molecule Imaging: Super-resolution microscopy combined with specific SYNCRIP antibodies could reveal detailed spatial organization of SYNCRIP-containing RNP complexes within cells, particularly during viral infection or cancer progression.

• CRISPR-based Approaches: CRISPR knock-in of epitope tags or fluorescent proteins at the endogenous SYNCRIP locus could enable antibody-independent tracking of SYNCRIP, avoiding potential artifacts from overexpression systems.

• Combinatorial Antibody Approaches: Using multiple antibodies against different SYNCRIP epitopes in multiplexed imaging or sequential immunoprecipitation could provide more comprehensive information about SYNCRIP's dynamic interactions and modifications.

What are the key unresolved questions about SYNCRIP function that antibody-based approaches might help answer?

Several important questions remain about SYNCRIP function that could be addressed using antibody-based approaches:

• Post-translational Modifications: Development of modification-specific antibodies (phospho-SYNCRIP, methylated SYNCRIP, etc.) could help elucidate how SYNCRIP activity is regulated and how these modifications change during viral infection or cancer progression.

• Isoform-specific Functions: SYNCRIP has multiple isoforms, and isoform-specific antibodies could help determine their distinct roles in different cellular contexts.

• Dynamic Localization: How does SYNCRIP shuttling between different cellular compartments regulate its function? Time-resolved immunofluorescence studies using specific antibodies could help address this question.

• Target Selectivity: What determines SYNCRIP's binding specificity to different RNA targets in different contexts? Combining CLIP-seq with immunoprecipitation using anti-SYNCRIP antibodies could help create comprehensive maps of SYNCRIP-RNA interactions under various conditions.

• Therapeutic Potential: Could targeting SYNCRIP be a viable approach for anti-viral or anti-cancer therapies? Antibody-based studies of SYNCRIP function in disease models could help answer this question.