SC35 Antibody
Description
Introduction to SC35 Antibody
SC35 antibody is a monoclonal antibody widely used to study nuclear speckles (NS), subnuclear domains enriched in pre-mRNA splicing factors. Historically, SC35 was believed to target the splicing factor SRSF2 (Serine/arginine-rich splicing factor 2), but recent studies demonstrate it primarily recognizes SRRM2 (Serine/arginine repetitive matrix protein 2), a core scaffold protein essential for NS formation . This antibody has been instrumental in advancing our understanding of RNA splicing, transcriptional regulation, and nuclear organization.
Role in Nuclear Speckle Formation
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SRRM2 and SON Dependency: Depletion of SRRM2 and SON (a splicing cofactor) leads to near-complete dissolution of nuclear speckles, confirming their roles as NS scaffolds .
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SC35 Antibody Mischaracterization: SC35 antibody was raised against spliceosomal extracts and later identified to target SRRM2, not SRSF2 as previously assumed .
Splicing and Transcriptional Regulation
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U2AF-Independent Splicing: SC35 (protein) restores splicing in U2AF-depleted extracts by bridging U1 snRNP and pre-mRNA, enabling spliceosome assembly .
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Autoregulation: SC35 protein promotes splicing of its own mRNA, generating unstable isoforms to control expression levels .
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Transcriptional Elongation: SC35 interacts with RNA Pol II and CDK9 (P-TEFb kinase) to facilitate transcriptional elongation .
Disease and Cellular Stress
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DNA Damage Response: SC35 accumulates in nuclear blebs induced by PARP inhibition, linking NS dysfunction to genomic instability .
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Mitotic Localization: During telophase, SC35 forms cytoplasmic rings and colocalizes with β-catenin at the plasma membrane .
Applications in Research
Controversies and Revisions
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Target Redefinition: Early studies misattributed SC35’s target to SRSF2 due to cross-reactivity with SRRM2 .
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Molecular Weight Discrepancy: While initial reports described SC35 as a 35 kDa protein, its primary target SRRM2 is ~300 kDa .
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Species Specificity: SC35 antibody shows variable reactivity in non-mammalian systems (e.g., Drosophila) .
Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Loss-of-function mutations in SC35 and SC35-like (SCL) proteins lead to a range of developmental changes in plants, including serrated leaves, delayed flowering, shorter roots, and abnormal silique phyllotaxy. SC35 and SCL proteins play essential roles in pre-mRNA splicing. PMID: 28273088
Q&A
What applications is SC35 antibody validated for?
SC35 antibody products are commercially available for several laboratory applications, with varying specifications depending on the supplier:
| Application | Abbreviation | Commonly Available | Notes |
|---|---|---|---|
| Western Blotting | WB | Yes | Detects primarily SRSF7 at ~35kDa under standard conditions |
| Immunohistochemistry (paraffin) | IHC-p | Yes | Nuclear speckle pattern |
| Enzyme-Linked Immunosorbent Assay | ELISA | Yes | Available from multiple suppliers |
| Immunocytochemistry | ICC | Some products | Nuclear speckle localization |
| Immunofluorescence | IF | Some products | Most common for visualizing nuclear speckles |
| Immunohistochemistry (frozen) | IHC-fr | Some products | Alternative to paraffin sections |
Researchers should verify the validated applications for their specific antibody product and be aware of the cross-reactivity with both SRRM2 and SRSF7 when interpreting results .
What cellular localization pattern does SC35 antibody typically display?
SC35 antibody staining exhibits a distinct nuclear speckled pattern because its main target, SRRM2, sharply localizes to nuclear speckles (NS) . Nuclear speckles are subnuclear domains enriched in pre-mRNA splicing factors and other RNA processing machinery. The SC35 monoclonal antibody is one of the most frequently used reagents to locate nuclear speckles in immunofluorescence studies . This characteristic pattern has made it a standard marker for nuclear speckles despite the recent clarification of its actual target protein.
Which species does SC35 antibody typically react with?
Based on commercial antibody information, SC35 antibodies show varying reactivity profiles:
| Species | Common Abbreviation | Typical Reactivity |
|---|---|---|
| Human | Hu | Most products |
| Mouse | Ms | Most products |
| Rat | Rt | Some products |
| Chicken | Ck | Limited products |
| Pig | Pg | Limited products |
| Arabidopsis | Ar | Specialized plant research antibodies |
Species reactivity varies between suppliers and specific products, so researchers should consult product-specific documentation before use in cross-species applications .
How was the misidentification of SC35 antibody's target discovered?
The misidentification was uncovered through a series of complementary experimental approaches:
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Immunoprecipitation-Mass Spectrometry (IP-MS): Analysis of proteins immunoprecipitated from HAP1 cell extracts using SC35 mAb revealed SRRM2 as the most significantly enriched protein, far exceeding any SR proteins including SRSF2 .
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Systematic SR-protein analysis: When all 12 canonical human SR-proteins were cloned into expression plasmids and purified using stringent conditions, immunoblotting revealed that among SR proteins, SC35 mAb primarily recognized SRSF7, not SRSF2 .
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Direct immunoprecipitation from whole-cell extracts: Using HEK293 cells, researchers observed clear enrichment of SRRM2 and SRSF7, but not SRSF2 or other factors in SC35 mAb immunoprecipitates .
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SRRM2 truncation experiments: Cell lines expressing truncated versions of SRRM2 demonstrated that SC35 mAb specifically recognizes epitopes in SRRM2, with signal remaining stable until specific portions of the C-terminal region were removed .
These multiple lines of evidence collectively proved that the primary target of SC35 mAb is SRRM2, with additional cross-reactivity to SRSF7.
What epitope regions of SRRM2 are recognized by SC35 monoclonal antibody?
Truncation experiments with SRRM2 have identified the epitope region recognized by SC35 monoclonal antibody:
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The signal from SC35 mAb remains relatively stable when up to 868 amino acids are removed from the SRRM2 C-terminus .
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The signal is reduced when 1,014 amino acids are removed and becomes completely undetectable with further truncation .
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This evidence suggests the epitope recognized by SC35 mAb is located in a specific region between amino acids 1,738 and 1,884 of the full-length SRRM2 protein (2,752 amino acids, based on sequence Q9UQ35-1) .
This region likely contains structural or sequence similarities to portions of SRSF7, explaining the cross-reactivity observed in immunoblotting experiments.
How does the cross-reactivity of SC35 mAb affect experimental design and data interpretation?
The cross-reactivity of SC35 mAb with both SRRM2 and SRSF7 requires careful experimental design and interpretation:
| Application | Primary Target | Potential Confounding Factors | Recommended Approach |
|---|---|---|---|
| Standard Western Blot | SRSF7 (~35 kDa) | May miss SRRM2 (~300 kDa) | Use gradient gels for SRRM2 detection |
| Gradient Gel Western Blot | SRRM2 | Standard transfer may be inefficient | Extended transfer times needed |
| Immunofluorescence | SRRM2 | Speckled pattern similar to other NS proteins | Co-staining with other markers |
| Immunoprecipitation | SRRM2 (primary) SRSF7 (secondary) | Complex mixtures of interacting proteins | Validation by mass spectrometry |
Studies using SC35 mAb should include controls with SRRM2 and/or SRSF7 knockdown/knockout to determine which protein is being detected in the specific experimental context .
What implications does the SRRM2 vs. SRSF2 targeting have for interpreting historical studies?
The discovery that SC35 mAb primarily targets SRRM2 rather than SRSF2 requires reevaluation of historical studies:
What controls are essential when using SC35 antibody in nuclear speckle research?
When using SC35 antibody for nuclear speckle research, several controls are essential:
| Control Type | Specific Control | Purpose |
|---|---|---|
| Molecular Identity | SRRM2 knockout/knockdown | Confirm SRRM2 contribution to staining pattern |
| SRSF7 knockout/knockdown | Assess SRSF7 contribution | |
| Co-staining with other NS markers (e.g., SON) | Validate nuclear speckle identification | |
| Technical | Secondary antibody-only control | Exclude non-specific binding |
| Isotype control antibody | Identify primary antibody background | |
| Gradient gels for Western blotting | Enable detection of high-MW SRRM2 | |
| Functional | Transcription inhibition (e.g., DRB) | Should cause speckle enlargement |
| Splicing inhibitors | Should reorganize nuclear speckles | |
| Combined SRRM2/SON depletion | Should disrupt nuclear speckles |
These controls ensure that the nuclear speckle structures identified by SC35 antibody staining are properly characterized and that experimental manipulations affecting these structures are correctly interpreted .
How do traditional immunoblotting techniques affect SC35 antibody target detection?
Traditional immunoblotting techniques have contributed to the historical misidentification of SC35 antibody targets:
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Standard SDS-PAGE gels (10-12%) efficiently resolve proteins in the 35-40 kDa range, leading to clear detection of SRSF7 (~35 kDa) .
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High molecular weight proteins like SRRM2 (~300 kDa) are poorly resolved and inefficiently transferred to membranes under standard conditions .
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The original study describing SC35 mAb reported a single 35 kDa band, which was likely SRSF7 rather than SRSF2 .
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Alternative techniques such as gradient gels (3-8% or 4-12%), extended transfer times, and specialized buffers are required to effectively detect SRRM2 by western blotting .
This technical limitation, combined with cross-reactivity to a protein of similar size to SRSF2, contributed to the persistent misidentification of the SC35 antibody's primary target for over two decades .
What alternative approaches should researchers consider for specific detection of SRSF2?
To specifically detect SRSF2 without the cross-reactivity issues of SC35 mAb:
| Approach | Methodology | Advantages | Limitations |
|---|---|---|---|
| Alternative antibodies | Use SRSF2-specific antibodies validated against knockouts | Direct detection | Requires thorough validation |
| Epitope tagging | Express tagged SRSF2 in experimental systems | High specificity | Potential functional interference |
| RNA-protein interaction | CLIP-seq for SRSF2-bound RNAs | Functional insights | Technically challenging |
| Custom antibodies | Generate against unique SRSF2 regions | Target specificity | Development time and cost |
| Combination approaches | Verify with multiple independent methods | Highest confidence | Resource intensive |
When selecting alternative approaches, researchers should require evidence of specificity through rigorous validation methods tailored to their experimental system.
How does SC35 antibody contribute to our understanding of nuclear speckle formation?
Recent research using SC35 antibody has revealed crucial insights about nuclear speckle formation:
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Nuclear speckles are formed primarily by two essential proteins: SON and SRRM2 (the main target of SC35 antibody) .
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Depletion of SON alone leads only to partial disassembly of nuclear speckles .
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Combined depletion of SON together with SRRM2 leads to near-complete dissolution of nuclear speckles, indicating their cooperative role in maintaining these structures .
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Genetic deletion of the intrinsically disordered regions of SRRM2, combined with SON depletion, also disrupts nuclear speckles .
These findings demonstrate that what researchers have been visualizing with SC35 antibody is primarily SRRM2, which along with SON forms the core structural components of nuclear speckles. This has significantly advanced our understanding of nuclear organization and RNA processing compartments .
Best practices for researchers using SC35 antibody
Based on current understanding of SC35 antibody specificity, researchers should:
