35 Antibody

What is SC-35 antibody and what cellular components does it recognize?

The antibody was raised against biochemically purified spliceosomes and has become one of the most frequently used reagents to locate nuclear speckles (NS) . Despite targeting primarily SRRM2, it also shows some reactivity with other SR proteins, particularly SRSF7, but not significantly with SRSF2 as originally believed .

For other 35-related antibodies, p-SC35 Antibody (SC-35) is a mouse monoclonal IgG1 kappa light chain antibody that detects p-SC35 in mouse, rat, and human samples through applications such as western blotting, immunoprecipitation, immunofluorescence, and immunohistochemistry .

What applications is SC-35 antibody validated for, and what are the recommended working dilutions?

SC-35 antibody has been validated for multiple research applications:

• Immunocytochemistry (ICC): SC-35 is widely used to visualize nuclear speckles in fixed cells

• Western Blotting (WB): Typically used at dilutions between 1:500-1:2000

• Immunoprecipitation (IP): For pulling down spliceosome components

• Immunofluorescence (IF): Used to study the distribution of splicing factors

• Immunohistochemistry (IHC): Both on paraffin-embedded (IHC-P) and frozen tissue sections

The antibody is available in various forms, including:

• Non-conjugated

• Agarose-conjugated for IP applications

• Fluorophore-conjugated versions (FITC, PE, and multiple Alexa Fluor conjugates) for direct immunofluorescence

When designing experiments, researchers should perform titration experiments to determine the optimal working concentration for their specific application and sample type.

How should researchers properly store and handle SC-35 antibody to maintain its activity?

Proper storage and handling of SC-35 antibody are essential for maintaining its specificity and activity:

• Long-term storage: Store at -20°C for up to one year

• Short-term/frequent use: Store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles: These can significantly degrade antibody quality and reduce binding affinity

Most commercial SC-35 antibodies are provided in a buffer containing:

• PBS as the base buffer

• 50% glycerol as a cryoprotectant

• 0.5% BSA as a stabilizer

• 0.02% sodium azide as a preservative

For long-term projects, consider aliquoting the antibody into single-use volumes to minimize freeze-thaw cycles and contamination risks.

What controls should researchers include when using SC-35 antibody in their experiments?

When using SC-35 antibody, proper experimental controls are critical for result interpretation and troubleshooting:

For Western Blotting:

• Positive control: Cell line/tissue known to express SRRM2

• Negative control: SRRM2 knockout/knockdown sample

• Loading control: Housekeeping protein to normalize expression levels

For Immunoprecipitation:

• Input control: Sample of the whole lysate (5-10% of amount used for IP)

• Isotype control: Matched IgG subclass (e.g., Mouse IgG1 for SC-35 monoclonal)

• Bead-only control: Beads added to lysate without antibody

For Immunofluorescence:

• Primary antibody omission control: To assess secondary antibody specificity

• Isotype control: To evaluate non-specific binding

• Competitive blocking: Pre-incubation with the immunizing peptide (if available)

• Ideally, SRRM2 knockout cells as negative control

A standardized validation approach defines a high-performing antibody as one that:

• In Western blot: Specifically detects the target in wild-type but not knockout samples

• In immunoprecipitation: Captures at least 10% of the target protein from starting material

• In immunofluorescence: Generates a signal at least 1.5-fold higher in WT versus KO cells

How has our understanding of SC-35 antibody specificity evolved, and what does this teach us about antibody validation?

The evolution of our understanding of SC-35 antibody specificity represents an important case study in antibody validation:

Originally developed in the 1990s against purified spliceosomes, SC-35 antibody was reported to recognize a 35 kDa protein identified as SRSF2 (previously known as SC-35) . For decades, it was widely used as a marker for this splicing factor.

• SC-35 monoclonal antibody primarily recognizes SRRM2, a much larger (~300 kDa) spliceosome-associated protein

• In immunoprecipitation experiments analyzed by mass spectrometry, SRRM2 was identified as the top target by a significant margin, with SR proteins having much lower scores

• When immunoprecipitates were analyzed by immunoblotting, researchers observed "a very clear enrichment for SRRM2 and SRSF7, but not for SRSF2, SRSF1 or other factors"

• Further validation using tagged and truncated SRRM2 constructs confirmed that SC-35 antibody recognizes SRRM2 on immunoblots, with the epitope appearing to be located between amino acids 868 and 1,014 from the C-terminus

The case of SC-35 highlights several critical lessons for antibody validation:

• Antibody targets should be regularly re-evaluated as technology advances

• Multiple validation methods should be employed (IP-MS, knockout controls, etc.)

• The cellular context (fixation, protein complexes) can influence epitope accessibility

• Original characterizations from decades ago may not have benefited from modern proteomics techniques

This evolution emphasizes why researchers should perform their own validation experiments rather than relying solely on historical data or manufacturer claims.

What are the methodological considerations for designing immunoprecipitation experiments with SC-35 antibody?

Successful immunoprecipitation with SC-35 antibody requires careful experimental design:

1. Antibody Selection and Preparation:

• For standard IP, use 2 μg of antibody per 500 μL of lysis buffer

• For mouse monoclonal antibodies like SC-35, use Protein G-conjugated beads (30μL of Dynabeads protein G)

• Consider using agarose-conjugated SC-35 antibody for direct IP without secondary capture

2. Lysis Buffer Selection:

• Choose buffers that preserve protein-protein interactions for co-IP applications

• Common options include Pierce IP Lysis Buffer or RIPA buffer with protease/phosphatase inhibitors

• For nuclear proteins like SRRM2, ensure efficient nuclear lysis (may require sonication)

3. Bead Selection Considerations:

4. Critical Protocol Steps:

• Pre-clear lysates with beads alone to reduce non-specific binding

• Rock antibody-bead conjugates for ~1 hour at 4°C

• Wash thoroughly to remove non-specifically bound proteins

• Remove supernatant by pipetting rather than aspiration to avoid bead loss

• Elute under appropriate conditions (reducing buffer for WB, mild conditions for functional studies)

5. Required Controls:

• Input control: To verify protein presence in starting material

• Isotype control: Matched IgG subclass (Mouse IgG1 for SC-35)

• Bead-only control: To identify non-specific bead interactions

6. Special Considerations for Nuclear Proteins:

• Ensure efficient nuclear lysis (may require optimization)

• Consider crosslinking for transient interactions

• Include RNase treatment controls if RNA-mediated interactions are suspected

Following these methodological considerations will maximize the specificity and efficiency of SC-35 antibody immunoprecipitation experiments.

What computational approaches are being developed for designing antibodies with customized specificity profiles?

Recent advances in computational antibody design represent a significant shift from traditional methods:

1. Biophysics-Informed Modeling Approach:

• Models are trained on experimentally selected antibodies

• Each potential ligand is associated with a distinct binding mode

• This enables prediction and generation of specific variants beyond those observed experimentally

2. Methodology and Workflow:

• Conduct phage display experiments with antibody libraries against various ligand combinations

• Build computational models using this training data

• Use models to predict outcomes for new ligand combinations

• Generate novel antibody sequences with predefined binding profiles

3. Specificity Design Strategies:

• For cross-specific sequences (binding multiple ligands): Jointly minimize energy functions associated with desired ligands

• For highly specific sequences (binding single ligand): Minimize energy function for desired ligand while maximizing for undesired ligands

4. Experimental Validation Process:

• Generate predicted antibody variants not present in initial libraries

• Test binding against target ligands to confirm specificity profiles

• Iterate on computational models based on experimental results

5. Advantages Over Traditional Methods:

• Overcomes library size limitations of experimental selection

• Provides greater control over specificity profiles

• Can differentiate between very similar epitopes

• Reduces experimental artifacts and biases

As stated in research findings: "This approach has applications for creating antibodies with both specific and cross-specific binding properties and for mitigating experimental artifacts and biases in selection experiments. The combination of biophysics-informed modeling and extensive selection experiments holds broad applicability beyond antibodies, offering a powerful toolset for designing proteins with desired physical properties."

How can active learning approaches optimize experimental resources in antibody research?

Active learning represents a significant advancement for resource-efficient antibody research:

1. Fundamental Concept:

• Start with a small labeled subset of data

• Iteratively expand the labeled dataset in an intelligent manner

• Prioritize experiments that maximize information gain

2. Application to Antibody-Antigen Binding Prediction:

• Library-on-library approaches generate many-to-many relationships between antibodies and antigens

• Machine learning models predict binding based on these relationships

• Active learning identifies which experiments to perform next for maximum model improvement

3. Performance Metrics from Research:

• Reduction in required antigen mutant variants: up to 35%

• Learning process acceleration: 28 steps faster than random baseline

• Three of fourteen tested algorithms significantly outperformed random sampling

4. Practical Implementation Strategy:

• Start with small initial screening of antibody-antigen pairs

• Build preliminary prediction model

• Use active learning algorithm to select next batch of experiments

• Update model with new data

• Repeat until desired prediction accuracy is achieved

5. Specific Value for Out-of-Distribution Prediction:

• Particularly valuable when test antibodies and antigens aren't represented in training data

• Helps bridge gaps between known and novel binding relationships

• Reduces experimental burden for exploring new antibody applications

These approaches are especially valuable for researchers working with specialized antibodies like SC-35, where experimental validation can be resource-intensive and time-consuming.

What methods should researchers use to validate antibody specificity when working with SC-35 or similar antibodies?

Comprehensive antibody validation is essential for research reproducibility. For SC-35 and similar antibodies, employ these methodological approaches:

1. Genetic Validation Strategies:

• Knockout/Knockdown Controls: Compare signals between wild-type and SRRM2 knockout/knockdown cells

• Overexpression Systems: Test antibody response to increasing levels of target protein

• Tagged Protein Expression: Create epitope-tagged versions of target protein for parallel detection

2. Biochemical Validation Methods:

• Western Blot: Confirm single band of expected molecular weight (note that SRRM2 is ~300kDa)

• Immunoprecipitation-Mass Spectrometry: Identify all proteins pulled down by the antibody

• Epitope Mapping: Use truncation constructs to identify the specific recognized region

3. Standardized Validation Criteria:
For an antibody to be considered high-performing:

• Western Blot: Must specifically detect target in WT but not KO lysate

• Immunoprecipitation: Must capture ≥10% of target from starting material

• Immunofluorescence: Must generate signal ≥1.5-fold higher in WT vs. KO cells

4. Advanced Validation Approaches:

• Orthogonal Targeting: Validate with multiple antibodies recognizing different epitopes

• Independent Detection Methods: Correlate antibody signal with mRNA levels or mass spectrometry data

• Mosaic Imaging Strategy: Plate WT and KO cells together to reduce staining/imaging bias

5. Tissue-Specific Validation:

• Tissue Panel Testing: Compare staining patterns across tissues with known expression profiles

• Cell Type-Specific Controls: Identify cell populations with varying target expression levels

• Fixation Method Comparison: Test antibody performance under different fixation conditions

For SC-35 specifically, the finding that it primarily recognizes SRRM2 rather than SRSF2 highlights the importance of thorough validation even for well-established antibodies . Researchers should consider re-validating historically used antibodies with modern techniques.

How should researchers design multiplexed experiments involving SC-35 antibody for co-localization studies?

Multiplexed immunofluorescence experiments with SC-35 antibody require careful design to avoid cross-reactivity and ensure reliable results:

1. Strategic Primary Antibody Selection:

• Host Species Combination: Use antibodies raised in different species (e.g., SC-35 mouse monoclonal with rabbit antibodies)

• Isotype Diversity: When using multiple mouse antibodies, select different isotypes (IgG1, IgG2a, etc.)

• Direct Conjugation Options: Consider using SC-35 directly conjugated to fluorophores (available in FITC, PE, and multiple Alexa Fluor conjugates)

2. Secondary Antibody Considerations:

• Cross-Adsorption: Use highly cross-adsorbed secondary antibodies to prevent cross-reactivity

• Specific subclass detection: For mouse antibodies, use isotype-specific secondaries (anti-mouse IgG1 for SC-35)

• Fluorophore Selection: Choose fluorophores with minimal spectral overlap

• Sequential Application: Consider sequential rather than simultaneous application

3. Validation Controls for Multiplexing:

• Single-Color Controls: Stain with each primary-secondary pair alone

• Fluorophore Minus One (FMO): Omit one fluorophore at a time to assess bleed-through

• Secondary-Only Controls: Apply secondary antibodies without primaries

• Absorption Controls: Pre-incubate primary with antigen when available

4. Advanced Multiplexing Strategies:

• Tyramide Signal Amplification (TSA): Allows use of same species antibodies

• Sequential Staining: Apply, image, and strip/quench antibodies in rounds

• Direct Conjugation: Use directly labeled primary antibodies

• Spectral Unmixing: Use spectral detectors and computational unmixing

5. Special Considerations for Nuclear Proteins:

• Nuclear Counterstain Selection: Choose nuclear stains compatible with nuclear speckle visualization

• Z-stack Acquisition: Capture full nuclear volume to assess true co-localization

• Super-Resolution Techniques: Consider STED or STORM for resolving subnuclear structures

Using these methodological approaches will ensure high-quality multiplexed imaging results when studying nuclear speckles and splicing factors with SC-35 antibody.

What troubleshooting approaches should be employed when SC-35 antibody yields inconsistent results?

When SC-35 antibody produces inconsistent results, a systematic troubleshooting approach is essential:

1. Antibody-Related Variables:

• Storage Conditions: Confirm proper storage at -20°C for long-term or 4°C for short-term use

• Freeze-Thaw Cycles: Excessive cycles can degrade antibody quality; use fresh aliquots

• Lot-to-Lot Variation: Compare performance between different antibody lots

• Concentration Optimization: Perform titration experiments to determine optimal working dilution

• Cross-Reactivity Assessment: Test for non-specific binding against similar proteins

2. Sample Preparation Factors:

• Fixation Methods: Different fixation protocols can affect epitope accessibility:

  • Paraformaldehyde: Preserves structure but may mask epitopes

  • Methanol: Better for some nuclear antigens but disrupts structure

  • Acetone: Can improve accessibility of certain epitopes

• Permeabilization Optimization: For nuclear proteins like SRRM2, sufficient permeabilization is crucial

• Antigen Retrieval: For fixed tissues, try heat-induced or enzymatic epitope retrieval methods

• Blocking Optimization: Test different blocking agents (BSA, serum, commercial blockers)

3. Protocol Modifications for Nuclear Targets:

• Nuclear Extraction Efficiency: Ensure complete nuclear lysis for western blotting and IP

• Chromatin State: Consider the impact of chromatin compaction on epitope accessibility

• Cell Cycle Variations: SRRM2/SC-35 distribution changes throughout cell cycle

4. Application-Specific Troubleshooting:

• Western Blotting:

  • Try different transfer methods for high molecular weight SRRM2 (~300kDa)

  • Optimize SDS-PAGE separation for clear resolution

  • Consider longer exposure times for weak signals

• Immunofluorescence:

  • Adjust detergent concentration for nuclear penetration

  • Try different mounting media to preserve fluorescence

  • Optimize imaging parameters for nuclear speckle visualization

• Immunoprecipitation:

  • Test different lysis buffers to maintain protein interactions

  • Increase antibody amount for low-abundance targets

  • Optimize wash stringency to balance specificity and yield

5. Controls for Validating Results:

• Positive and Negative Controls: Include known positive and negative samples

• Alternative Antibodies: Test different antibodies against the same target

• Genetic Validation: Use SRRM2 knockout/knockdown controls when available

When troubleshooting SC-35 antibody specifically, remember that its primary target is SRRM2 rather than SRSF2 as originally thought , which may explain some experimental inconsistencies observed in historical literature.

How can researchers incorporate SC-35 antibody in studies of disease-related splicing dysregulation?

SC-35 antibody can be a valuable tool for investigating splicing dysregulation in disease contexts:

1. Methodological Approaches for Disease-Related Studies:

• Nuclear Speckle Morphology Analysis:

  • Quantify changes in size, number, and intensity of SC-35-positive speckles

  • Compare patterns between normal and pathological tissues

  • Correlate alterations with disease progression markers

• Co-Localization with Disease-Associated Splicing Factors:

  • Perform multiplexed staining with SC-35 and disease-relevant proteins

  • Quantify co-localization coefficients using appropriate algorithms

  • Track changes in spatial relationships during disease development

2. Applications in Cancer Research:

• Diagnostic Marker Development:

  • Evaluate SC-35 staining patterns across tumor grades/stages

  • Determine prognostic value of nuclear speckle alterations

  • Develop scoring systems for pathology applications

• Therapeutic Response Monitoring:

  • Assess changes in splicing factor distribution following treatment

  • Correlate nuclear speckle reorganization with treatment efficacy

  • Identify resistant phenotypes based on splicing patterns

3. Neurodegenerative Disease Applications:

• Protein Aggregation Studies:

  • Investigate co-localization of SC-35 with disease-associated aggregates

  • Examine sequestration of splicing factors in inclusion bodies

  • Track temporal changes in nuclear organization during disease progression

4. Advanced Techniques for Mechanistic Insights:

• Live-Cell Imaging:

  • Use fluorescently-tagged SC-35 antibody fragments for dynamic studies

  • Monitor real-time changes in nuclear speckle behavior

  • Correlate with functional readouts of splicing activity

• Proximity Ligation Assay (PLA):

  • Detect protein-protein interactions between SRRM2 and disease-relevant factors

  • Quantify interaction changes during disease progression

  • Map spatial distribution of interactions within the nucleus

• CLIP-Seq Integration:

  • Combine SC-35 immunoprecipitation with RNA sequencing

  • Identify differentially bound transcripts in disease states

  • Correlate with alternative splicing patterns

5. Experimental Design for Translational Research:

• Patient-Derived Models:

  • Apply SC-35 staining to patient-derived organoids or xenografts

  • Compare patterns with primary patient samples

  • Validate findings across multiple patient cohorts

• High-Throughput Screening Approaches:

  • Develop automated image analysis pipelines for SC-35 pattern recognition

  • Screen compound libraries for agents that normalize disrupted patterns

  • Identify novel therapeutic targets in the splicing machinery

When using SC-35 antibody in disease-related research, researchers should remember that it primarily recognizes SRRM2 , which may offer new interpretations of historical findings about splicing dysregulation in various pathologies.