scoca Antibody

What is the SC35 antibody and what cellular structures does it primarily detect?

SC35 antibody (clone SC-35) is a mouse monoclonal antibody that functions as a nuclear speckle marker, recognizing the serine/arginine-rich splicing factor 2 (SRSF2/SFRS2). This antibody specifically targets SC35, a protein involved in pre-mRNA splicing that localizes to nuclear speckles (NS). SC35 is required for the formation of the earliest ATP-dependent splicing complex and interacts with spliceosomal components during spliceosome assembly . The antibody allows researchers to visualize these dynamic subnuclear structures that serve as storage and assembly sites for splicing factors.

What are the species reactivity profiles for commercially available SC35 antibodies?

The SC35 antibody clone SC-35 has been validated for human, mouse, and rat samples. This specific clone is the most widely used for SC35 detection in the research market and has been cited in over 135 publications, indicating its reliability and broad acceptance in the scientific community . Species reactivity is a critical consideration when designing experiments, as antibodies may show variable specificity across different organisms due to epitope conservation differences.

What are the primary applications of SC35 antibody in cellular research?

SC35 antibody is predominantly used in immunocytochemistry/immunofluorescence (ICC/IF) applications to visualize nuclear speckles. These applications are particularly valuable for:

• Studying RNA processing and splicing mechanisms

• Investigating nuclear organization during normal cellular function

• Examining changes in nuclear architecture during viral infections, particularly HIV-1

• Serving as a control marker for nuclear speckle compartments in colocalization studies

What are the optimal fixation and permeabilization methods for SC35 immunofluorescence staining?

For optimal SC35 immunofluorescence staining, researchers should follow these methodological steps:

• Fix cells with 2-4% paraformaldehyde (PFA) for 5-10 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

• Block with 3% BSA in PBS for 1 hour at room temperature

• Incubate with primary SC35 antibody (typically at 1:1000-1:3000 dilution) overnight at 4°C

• Wash with PBST (PBS + 0.1% Tween-20)

• Incubate with appropriate secondary antibody (e.g., goat anti-mouse-Alexa Fluor 488/594/Cy5) at 1:1000-1:2000 dilution

• Counterstain nuclei with DAPI or Hoechst 33342

This protocol yields consistent staining of nuclear speckles with minimal background, facilitating clear visualization of these subnuclear structures.

How can SC35 antibody be effectively utilized in multicolor immunofluorescence experiments?

For multicolor immunofluorescence using SC35 antibody:

• Antibody selection and compatibility: Choose primary antibodies raised in different host species (e.g., mouse anti-SC35 paired with rabbit anti-protein of interest) to avoid cross-reactivity

• Fluorophore selection: Select fluorophores with minimal spectral overlap (e.g., Alexa Fluor 488, 568, and 647)

• Sequential staining approach:

  • For co-staining with other mouse antibodies, use Zenon labeling kits for direct conjugation

  • Alternatively, apply sequential staining with complete blocking between antibody sets

• Controls: Include single-stained samples for compensation controls

• Image acquisition parameters: Capture each channel separately with appropriate filter sets to prevent bleed-through

This approach has been successfully used in HIV-1 studies to simultaneously visualize SC35-marked nuclear speckles, HA-tagged CPSF6, and INmNG-tagged HIV-1 viral components .

What concentrations and dilution factors are recommended for SC35 antibody in different experimental setups?

Based on published protocols, these are recommended working dilutions for SC35 antibody:

Optimization may be necessary depending on specific cell types, fixation methods, and detection systems used.

How is SC35 antibody used to study HIV-1 nuclear entry and viral DNA integration?

SC35 antibody serves as a crucial tool for studying HIV-1 nuclear entry and integration by:

• Identifying nuclear speckles as viral interaction sites: SC35 staining allows visualization of nuclear speckles where HIV-1 forms membraneless organelles (HIV-1-MLOs) after nuclear entry

• Analyzing colocalization patterns: Researchers use SC35 antibody alongside viral markers to quantify the percentage of HIV-1 components that associate with nuclear speckles

• Temporal tracking of infection: By fixing cells at different time points post-infection (e.g., 8 hpi, 16 hpi) and staining with SC35, researchers can track the progression of HIV-1 nuclear entry

• Evaluating host factor dependencies: SC35 staining helps evaluate how knockdown of factors like SON and SRRM2 affects HIV-1 localization to nuclear speckles

This methodology has revealed that HIV-1 forms nuclear condensates that safeguard the virus against cellular immune sensors like cGAS, providing a mechanism for immune evasion .

What role does SC35 antibody play in investigating the relationship between nuclear speckles and HIV-1 capsid interactions?

SC35 antibody enables researchers to:

• Map spatial relationships: Determine the precise spatial relationship between HIV-1 capsid proteins and nuclear speckle components

• Quantify colocalization: Assess what percentage of HIV-1 capsids localize to SC35-positive nuclear speckles during infection

• Track kinetics of association: Monitor how quickly and to what extent HIV-1 components associate with nuclear speckles after nuclear entry

• Evaluate impact of interventions: Test how drugs like PF74 (which disrupts CypA-capsid interactions) affect the association between HIV-1 and nuclear speckles

• Analyze host factor requirements: Determine which host factors (e.g., CPSF6) are essential for HIV-1 localization to nuclear speckles

Research using these approaches has demonstrated that HIV-1 capsids associate with SC35-positive nuclear speckles within 8 hours post-infection, and this association is dependent on the host factor CPSF6 .

How can SC35 antibody staining be quantitatively analyzed to assess nuclear speckle dynamics?

Quantitative analysis of SC35 antibody staining can be performed through:

• Intensity measurements:

  • Measure SC35 signal intensity within defined nuclear regions

  • Quantify changes in total fluorescence intensity across conditions

  • Compare SC35 intensities within 10 nuclear speckles per cell across multiple cells (n=10 cells, 100 total NSs per condition)

• Morphological analysis:

  • Measure size, number, and distribution of SC35-positive speckles

  • Track changes in speckle properties during cell cycle or stress conditions

  • Analyze speckle fusion/fission events over time

• Colocalization analysis:

  • Calculate Pearson's or Mander's coefficients between SC35 and proteins of interest

  • Determine percentage of overlapping volume between SC35 and viral components

  • Establish spatial relationships with distance measurements from nuclear speckles

• Advanced imaging techniques:

  • Apply deconvolution to remove out-of-focus light (using software like LASX)

  • Implement machine learning algorithms for automated detection and classification

  • Utilize 3D reconstruction for volumetric analysis of nuclear speckles

These quantitative approaches have been instrumental in demonstrating that HIV-1 preferentially associates with nuclear speckles during infection, providing insights into viral replication strategies .

What are common issues with SC35 antibody staining and how can they be resolved?

Common issues and their solutions include:

• High background or non-specific staining:

  • Increase blocking time (3% BSA for 1-2 hours)

  • Optimize antibody concentration (try higher dilutions)

  • Include additional wash steps with 0.1% Triton X-100

  • Pre-absorb secondary antibodies with cell lysate

• Weak or inconsistent nuclear speckle signals:

  • Ensure proper fixation (fresh 4% PFA)

  • Optimize permeabilization (0.1-0.5% Triton X-100)

  • Try different antibody incubation times/temperatures

  • Use signal amplification systems if necessary

• Difficulty distinguishing nuclear speckles in double knockdown experiments:

  • Use intensity scaling to exaggerate nuclear speckle signal

  • Apply deconvolution algorithms to improve signal-to-noise ratio

  • Implement reference markers to identify nuclear speckle regions

• Batch-to-batch variability:

  • Use consistent antibody lots when possible

  • Include positive controls in each experiment

  • Normalize data to internal standards

How can researchers validate the specificity of SC35 antibody staining patterns?

To validate SC35 antibody specificity:

• Genetic approaches:

  • Perform siRNA/shRNA knockdown of SC35/SRSF2 to confirm signal reduction

  • Use CRISPR-Cas9 SC35 knockout cells as negative controls

  • Express tagged versions of SC35 and verify colocalization with antibody staining

• Biochemical validations:

  • Conduct western blotting to confirm antibody recognizes a protein of the correct size

  • Perform immunoprecipitation followed by mass spectrometry to verify target identity

  • Use peptide competition assays to demonstrate epitope-specific binding

• Imaging validations:

  • Compare staining patterns with multiple antibodies against SC35 from different sources

  • Use super-resolution microscopy to confirm expected nuclear speckle morphology

  • Perform colocalization with other known nuclear speckle markers like SON or SRRM2

• Functional validations:

  • Correlate SC35 staining patterns with known biological processes (e.g., changes during cell cycle)

  • Verify expected responses to transcriptional inhibitors or splicing modulators

What considerations should be made when selecting secondary antibodies for SC35 detection?

When selecting secondary antibodies for SC35 detection:

• Host species compatibility:

  • Choose secondary antibodies raised against the host species of the SC35 primary antibody (typically anti-mouse for clone SC-35)

  • Ensure secondary antibodies are highly cross-adsorbed to prevent cross-reactivity in multi-labeling experiments

• Fluorophore selection:

  • Consider the spectral properties of imaging systems available

  • Choose fluorophores that match filter sets and avoid autofluorescence ranges

  • For colocalization studies with INmNG-tagged HIV-1, goat anti-mouse-Cy5 has been successfully used

• Application-specific optimizations:

  • For confocal microscopy: bright, photostable fluorophores like Alexa Fluor 488, 568, or 647

  • For super-resolution: fluorophores with appropriate photophysical properties

  • For long-term imaging: fluorophores resistant to photobleaching

• Signal amplification needs:

  • Standard detection: directly conjugated secondaries at 1:1000-1:2000

  • Enhanced sensitivity: tyramide signal amplification or multi-layered detection systems

  • Low abundance targets: consider quantum dots or other high-brightness conjugates

How should researchers interpret changes in SC35 staining patterns during viral infection?

When interpreting SC35 staining pattern changes during viral infection:

• Morphological changes:

  • Enlargement of speckles often indicates increased splicing activity or stress response

  • Fragmentation may suggest disruption of splicing machinery by viral factors

  • Redistribution within the nucleus may indicate selective recruitment to viral replication sites

• Intensity alterations:

  • Increased SC35 intensity in specific nuclear regions suggests accumulation of splicing factors

  • Decreased global intensity might indicate degradation or masking of epitopes

  • Focal intense signals may represent sites of active viral transcription/replication

• Colocalization context:

  • High colocalization between SC35 and viral proteins suggests hijacking of splicing machinery

  • Formation of distinct SC35/viral protein condensates indicates establishment of viral factories

  • Partial overlap may represent transitional states during infection progression

• Temporal dynamics:

  • Early infection (8 hpi): Association of HIV-1 components with SC35-positive speckles indicates nuclear entry

  • Later timepoints (16 hpi): Formation of CPSF6 puncta with SC35 colocalization suggests establishment of HIV-1 membraneless organelles (HIV-1-MLOs)

What advanced imaging techniques can enhance SC35 antibody-based nuclear speckle research?

Advanced imaging techniques for SC35 research include:

• Super-resolution microscopy:

  • Structured Illumination Microscopy (SIM): Achieves ~100nm resolution, suitable for resolving internal structure of nuclear speckles

  • Stimulated Emission Depletion (STED): Provides ~30-50nm resolution for detailed speckle architecture

  • Single-Molecule Localization Microscopy (PALM/STORM): Enables molecular-scale mapping of SC35 distribution

• Live-cell imaging approaches:

  • Complement SC35 antibody studies with live-cell compatible tags (e.g., SC35-GFP)

  • Use lattice light-sheet microscopy for low-phototoxicity 3D imaging

  • Apply fluorescence recovery after photobleaching (FRAP) to assess SC35 dynamics

• Correlative microscopy:

  • Combine immunofluorescence with electron microscopy for ultrastructural context

  • Implement CLEM (Correlative Light and Electron Microscopy) for precise location of SC35 signals

• Computational image analysis:

  • Apply deconvolution algorithms to improve signal-to-noise ratio in widefield microscopy

  • Use machine learning for automated segmentation and classification of nuclear speckles

  • Implement 3D reconstruction and volumetric analysis to fully characterize speckle architecture

How can quantitative analysis of nuclear speckles contribute to understanding HIV-1 pathogenesis?

Quantitative analysis of nuclear speckles using SC35 antibody contributes to understanding HIV-1 pathogenesis by:

This quantitative approach has revealed that HIV-1 forms nuclear condensates that protect viral DNA from cellular immune sensors, providing a mechanism for immune evasion during infection .