SMARCC2 Antibody, FITC conjugated

What is SMARCC2 and what functional role does it play in chromatin remodeling?

SMARCC2 (also known as BAF170) is one of the core subunits of the SWI/SNF (Switch/Sucrose Non-Fermenting) chromatin remodeling complex, which is necessary for efficient nucleosome remodeling by Brg1 in vitro . This protein contains two key functional domains: SWIRM (Ile424-Thr521) and SANT (Ser596-Pro647) . The SWIRM domain, along with two RPT domains, forms part of the complex, while the SANT domain, together with the preHSA domain of SMARCC4 and the C-terminal helix of SMARCD1, composes the thumb part of the basic module of the SWI/SNF complex . SMARCC2 plays a crucial role in the early assembly of SWI/SNF complexes and has been implicated in transcriptional regulation through its ability to dynamically regulate chromatin structure .

How can researchers distinguish between SMARCC2 and other related SWI/SNF complex proteins?

Distinguishing SMARCC2 from other SWI/SNF complex proteins requires careful consideration of experimental parameters:

• Antibody specificity: Select antibodies that target unique epitopes specific to SMARCC2, such as those recognizing regions outside the conserved domains shared with related proteins like SMARCC1 .

• Molecular weight analysis: SMARCC2/BAF170 typically appears at 162-170 kDa on Western blots, which can help distinguish it from other SWI/SNF components .

• Differential expression patterns: Unlike some SWI/SNF components, SMARCC2/BAF170 is absent in pluripotent embryonic stem (ES) cells but is up-regulated in neurons/neuronal progenitors upon differentiation of mouse ES cells with retinoic acid .

• Co-immunoprecipitation studies: Computational analysis of IP-MS data has confirmed very strong competition between SMARCC1-SMARCC2 and SMARCD1-SMARCD2-SMARCD3, which can be leveraged to distinguish these closely related proteins .

What applications are FITC-conjugated SMARCC2 antibodies best suited for?

FITC-conjugated SMARCC2 antibodies are particularly valuable for the following applications:

• Immunofluorescence microscopy: The FITC conjugation enables direct visualization of SMARCC2 localization without requiring secondary antibodies, reducing background and cross-reactivity issues .

• Flow cytometry: FITC-conjugated antibodies allow for detection of SMARCC2 expression at the single-cell level, enabling researchers to quantify expression across heterogeneous cell populations .

• Chromatin immunoprecipitation (ChIP): While traditional ChIP often uses unconjugated antibodies, FITC-conjugated antibodies can be utilized in modified protocols to visualize chromatin-antibody complexes or in specialized ChIP variants .

• CUT&RUN assays: The fluorescent tag can assist in quality control steps when optimizing these advanced chromatin mapping techniques .

What are the optimal fixation and permeabilization protocols for immunofluorescence using FITC-conjugated SMARCC2 antibodies?

When designing immunofluorescence experiments with FITC-conjugated SMARCC2 antibodies, researchers should consider the following methodological approach:

• Fixation: A dual fixation protocol is recommended, starting with 10 minutes in 4% paraformaldehyde at room temperature followed by a brief (5-minute) post-fixation with ice-cold methanol. This combination preserves both protein epitopes and nuclear architecture .

• Permeabilization: Use 0.2% Triton X-100 in PBS for 10 minutes at room temperature after fixation. For highly sensitive applications, a gentler approach using 0.1% saponin may better preserve the fluorescence intensity of the FITC conjugate .

• Blocking: Implement a robust blocking step using 5% BSA and 5% normal serum (from the same species as the secondary antibody if using additional primary antibodies) for at least 1 hour to minimize background fluorescence, which is particularly important with directly conjugated antibodies .

• Nuclear counterstaining: Select DNA dyes with minimal spectral overlap with FITC (488nm excitation/520nm emission), such as DAPI or Hoechst, to facilitate clear visualization of nuclear SMARCC2 localization .

The dilution factor should be empirically determined, but starting with a 1:50 to 1:100 dilution is generally appropriate for most FITC-conjugated antibodies in immunofluorescence applications .

How should researchers optimize ChIP protocols when using SMARCC2 antibodies?

Optimizing ChIP protocols for SMARCC2 antibodies requires careful attention to several key methodological parameters:

For FITC-conjugated antibodies specifically in ChIP applications, researchers should ensure that the fluorophore doesn't interfere with epitope recognition by including additional validation steps comparing conjugated versus unconjugated antibody performance . Additionally, SimpleChIP Enzymatic Chromatin IP Kits have been validated for use with SMARCC2 antibodies and provide a standardized protocol that can be adapted for conjugated antibodies .

What controls are essential when validating SMARCC2 antibody specificity?

Rigorous validation of SMARCC2 antibody specificity requires multiple complementary approaches:

• Positive controls:

  • Western blotting using lysates from cell lines known to express high levels of SMARCC2 (avoid pluripotent embryonic stem cells which lack SMARCC2 expression)

  • Immunoprecipitation followed by mass spectrometry to confirm pull-down of known SMARCC2 interaction partners

• Negative controls:

  • CRISPR-Cas9 knockout validation using the approach described in the literature, where U118MG cells were transfected with sgRNA-SMARCC2 plasmid and single-cell cloned

  • Isotype control antibodies (rabbit IgG) processed in parallel to assess non-specific binding

  • Peptide competition assays where excess immunizing peptide blocks specific antibody binding

• Cross-reactivity assessment:

  • Testing against recombinant SMARCC1 protein (the closest paralog) to ensure specificity

  • Species cross-reactivity verification, as SMARCC2 antibodies have demonstrated reactivity with human, mouse, rat, and monkey samples

• Functional validation:

  • ChIP-qPCR at known SMARCC2 binding sites before proceeding to genome-wide assays

  • Correlation of immunofluorescence patterns with expected nuclear localization and exclusion from nucleoli

How can FITC-conjugated SMARCC2 antibodies be used to study SWI/SNF complex dynamics during cell differentiation?

FITC-conjugated SMARCC2 antibodies offer unique advantages for studying the dynamic assembly and disassembly of SWI/SNF complexes during cellular differentiation processes:

• Time-lapse microscopy: The direct fluorescent conjugation allows for live-cell imaging of SMARCC2 dynamics during differentiation without requiring cell fixation. This approach can capture transient interactions and redistribution patterns as cells transition between states .

• FACS-based differentiation tracking: Research has shown that SMARCC2/BAF170 expression is upregulated during neural differentiation of embryonic stem cells . FITC-conjugated antibodies enable researchers to isolate and characterize cells at different differentiation stages based on SMARCC2 expression levels using flow cytometry.

• Co-localization studies: By combining FITC-conjugated SMARCC2 antibodies with antibodies against other differentiation markers or chromatin regulators labeled with spectrally distinct fluorophores, researchers can analyze the temporal coordination of SWI/SNF complex assembly with other cellular processes.

• Methodological approach:

  • Induce differentiation using established protocols (e.g., retinoic acid treatment for neuronal differentiation)

  • Perform sequential sampling at defined timepoints

  • Process samples for either flow cytometry or immunofluorescence

  • Quantify SMARCC2 levels and nuclear distribution patterns

  • Correlate changes with differentiation markers and functional outcomes

This approach has revealed that exogenous expression of SMARCC2/BAF170 leads to loss of stem cell pluripotency and self-renewal, highlighting its crucial role in differentiation processes .

What methodological approaches allow researchers to investigate SMARCC2's role in transcriptional regulation?

To investigate SMARCC2's role in transcriptional regulation, researchers can employ several complementary methodological approaches:

• ChIP-seq with SMARCC2 antibodies: This approach identifies genome-wide binding sites of SMARCC2, revealing its association with specific regulatory elements. The recommended protocol involves:

  • Chromatin preparation from 4-6 × 10^6 cells using a 1:50 dilution of SMARCC2 antibody

  • Library preparation optimized for transcription factor binding sites

  • Bioinformatic analysis focusing on promoter regions and enhancers

• CUT&RUN assays: This technique offers higher signal-to-noise ratio than traditional ChIP and can be performed with fewer cells:

  • Follow established CUT&RUN protocols using a 1:50 dilution of SMARCC2 antibody

  • This approach has been validated with SMARCC2 antibodies using the CUT&RUN Assay Kit #86652

• Transcriptome analysis following SMARCC2 manipulation:

  • Generate SMARCC2 knockout or overexpression cell lines using CRISPR-Cas9 or lentiviral approaches as described in the literature

  • For knockout, use CRISPR-Cas9-SMARCC2 (sgSMARCC2) plasmid followed by single-cell clonal culture

  • For overexpression, use SMARCC2-overexpressing lentiviral vectors (oeSMARCC2) at 100 nM concentration

  • Perform RNA-seq to identify differentially expressed genes

• Mechanistic studies of target gene regulation:

  • Research has shown that SMARCC2 can negatively regulate transcription by dynamically altering chromatin structure at specific promoter regions, such as DKK1

  • This involves SMARCC2-mediated closure of promoter regions that can be bound by transcription factors like EGR1

These approaches collectively allow for a comprehensive investigation of how SMARCC2 contributes to transcriptional regulation through chromatin remodeling activities.

How do SMARCC2 antibodies help elucidate the composition and competition within BAF complexes?

SMARCC2 antibodies have been instrumental in revealing the complex subunit composition and competitive interactions within BAF chromatin remodeling complexes:

The resulting data provide important insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest potential approaches for therapeutically targeting specific BAF components in disease contexts .

How should researchers interpret different molecular weight bands when using SMARCC2 antibodies in Western blots?

When performing Western blot analysis with SMARCC2 antibodies, researchers may observe multiple bands that require careful interpretation:

• Expected band pattern:

  • The primary SMARCC2/BAF170 bands typically appear at 162 and 170 kDa

  • These represent different isoforms or post-translationally modified versions of the protein

• Methodological approach for accurate interpretation:

  • Run positive controls from cell lines with confirmed SMARCC2 expression

  • Include SMARCC2 knockout samples (generated using CRISPR-Cas9) as negative controls

  • Use a gradient gel (4-12%) to achieve optimal separation of high molecular weight proteins

  • For Western blotting, use a 1:1000 dilution of the primary antibody as recommended

• Interpretation of additional bands:

  • Lower molecular weight bands (~100-120 kDa): May represent proteolytic fragments or alternative splice variants

  • Higher molecular weight bands (>170 kDa): Could indicate post-translational modifications such as ubiquitination or SUMOylation

  • Bands that appear in both wildtype and knockout samples should be considered non-specific

• Verification strategies:

  • Peptide competition assays to confirm specificity of observed bands

  • Mass spectrometry analysis of excised gel bands to confirm protein identity

  • Comparison of band patterns across different antibodies targeting different SMARCC2 epitopes

This comprehensive approach ensures accurate interpretation of Western blot results when using SMARCC2 antibodies for protein detection and quantification.

What factors can affect the sensitivity and specificity of FITC-conjugated SMARCC2 antibodies in flow cytometry?

Several factors can significantly impact the performance of FITC-conjugated SMARCC2 antibodies in flow cytometry experiments:

• Fixation and permeabilization effects:

  • Overfixation with paraformaldehyde (>2%) can mask epitopes and reduce signal intensity

  • Insufficient permeabilization may prevent antibody access to nuclear SMARCC2

  • Recommended protocol: 2% paraformaldehyde for 10 minutes followed by permeabilization with 0.1% Triton X-100

• Fluorophore considerations:

  • FITC is susceptible to photobleaching, requiring samples to be protected from light during processing

  • FITC fluorescence is pH-sensitive, with optimal emission at pH 8.0; maintain appropriate buffer conditions

  • FITC signal can overlap with cellular autofluorescence, particularly in fixed cells

• Antibody titration importance:

  • Insufficient antibody concentration leads to weak signal

  • Excess antibody increases background and non-specific binding

  • Perform titration experiments (typically starting with 1:50 dilution) to determine optimal concentration

• Methodological optimization strategies:

  • Include a blocking step with 5% normal serum to reduce non-specific binding

  • Perform parallel staining with isotype control antibodies (rabbit IgG-FITC)

  • Include singly-stained controls for compensation when multiplexing with other fluorophores

  • Consider alternative fluorophores (e.g., Alexa Fluor 488) for applications requiring higher photostability

• Cell preparation considerations:

  • Single-cell suspensions are critical; incomplete dissociation causes aggregate artifacts

  • Dead cell exclusion dyes should be used and compatible with FITC detection

  • Nuclear integrity must be maintained during permeabilization for accurate SMARCC2 detection

By systematically addressing these factors, researchers can maximize both sensitivity and specificity when using FITC-conjugated SMARCC2 antibodies in flow cytometry applications.

How can researchers validate that SMARCC2 antibodies are detecting the intended target in complex experimental systems?

Comprehensive validation that SMARCC2 antibodies are detecting the intended target requires multiple orthogonal approaches:

• Genetic validation approaches:

  • CRISPR-Cas9 knockout: Follow established protocols using sgRNA-SMARCC2 plasmid transfection followed by single-cell clonal culture as described in the literature

  • siRNA knockdown: Use transient knockdown to confirm reduction in antibody signal correlates with reduced SMARCC2 mRNA levels

  • Overexpression: Transfect cells with SMARCC2-overexpressing lentiviral vectors and confirm increased antibody signal

• Biochemical validation techniques:

  • Immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein

  • Western blotting following immunoprecipitation to verify the correct molecular weight (162-170 kDa)

  • Peptide competition assays using the immunizing peptide (e.g., recombinant human probable global transcription activator SNF2L2 protein, 700-1216AA)

• Cross-platform validation:

  • Correlate protein detection across multiple techniques (Western blot, immunofluorescence, flow cytometry)

  • Compare results using antibodies targeting different epitopes of SMARCC2

  • Verify that the antibody detects expected changes in SMARCC2 levels during biological processes (e.g., upregulation during neuronal differentiation)

• Functional validation:

  • ChIP-seq or CUT&RUN experiments should show enrichment at genomic regions consistent with known SMARCC2 binding patterns

  • Knockdown/knockout followed by rescue experiments to demonstrate specificity of observed phenotypes

  • Co-immunoprecipitation should pull down known SMARCC2 interaction partners (e.g., other BAF complex components)

This multi-faceted validation approach provides robust evidence that the antibody is specifically detecting SMARCC2 rather than cross-reacting with other proteins or producing artifacts.

How can SMARCC2 antibodies be used to investigate its role in cancer progression?

Recent research has identified important connections between SMARCC2 and cancer biology that can be investigated using antibody-based approaches:

• Expression pattern analysis in tumor samples:

  • Immunohistochemistry using SMARCC2 antibodies has revealed that SMARCC2 expression is lower in malignant glioblastoma (GBM) compared to low-grade gliomas

  • This finding suggests its potential role as a tumor suppressor in specific cancer contexts

  • Researchers can employ SMARCC2 antibodies in tissue microarray analysis to evaluate expression across tumor grades and correlate with patient outcomes

• Mechanistic studies of tumor suppression:

  • SMARCC2 knockout has been shown to promote the proliferation of glioblastoma cells, while its overexpression inhibits proliferation

  • Researchers can use SMARCC2 antibodies in ChIP-seq experiments to identify direct regulatory targets that mediate this tumor-suppressive function

  • The DKK1 signaling axis has been identified as a key target, with SMARCC2 negatively regulating DKK1 transcription by dynamically regulating chromatin structure

• Signaling pathway investigation:

  • SMARCC2 antibodies can be used in co-immunoprecipitation experiments to identify interaction partners in cancer cells

  • Phospho-specific antibodies against AKT can be used in conjunction with SMARCC2 manipulation to validate the finding that DKK1 knockdown reduces glioblastoma cell proliferation by inhibiting the PI3K-AKT pathway

• Methodological approach for cancer studies:

  • Generate stable SMARCC2 knockout and overexpression cell lines using established protocols

  • Perform phenotypic assays (proliferation, migration, invasion) to assess functional consequences

  • Use SMARCC2 antibodies in ChIP experiments to map genomic binding sites in cancer versus normal cells

  • Correlate binding patterns with transcriptional outputs using RNA-seq

These approaches collectively provide a comprehensive framework for investigating SMARCC2's role in cancer progression using antibody-based techniques.

What novel applications are emerging for FITC-conjugated SMARCC2 antibodies in chromatin dynamics research?

Several innovative applications are emerging for FITC-conjugated SMARCC2 antibodies in the study of chromatin dynamics:

• Live-cell imaging of BAF complex assembly:

  • FITC-conjugated antibodies can be delivered into living cells using cell-penetrating peptides or microinjection

  • This approach enables real-time visualization of SMARCC2 recruitment to specific genomic loci during cellular responses to stimuli

  • When combined with fluorescently tagged chromatin markers, this technique provides insights into the kinetics of chromatin remodeling events

• Super-resolution microscopy applications:

  • FITC-conjugated SMARCC2 antibodies can be used in techniques like Structured Illumination Microscopy (SIM) or Stochastic Optical Reconstruction Microscopy (STORM)

  • These approaches overcome the diffraction limit of conventional microscopy, allowing visualization of subnuclear SMARCC2 distribution at nanometer resolution

  • This reveals previously undetectable patterns of BAF complex organization relative to chromatin domains

• CUT&RUN and CUT&Tag advancements:

  • FITC-conjugated antibodies can be used in modified CUT&RUN protocols that incorporate fluorescence-activated sorting of protein-DNA complexes

  • This enhances the signal-to-noise ratio and enables analysis from limited cell numbers

  • The CUT&RUN approach has been validated with SMARCC2 antibodies using a 1:50 dilution

• Single-cell epigenomic profiling:

  • FITC-conjugated SMARCC2 antibodies enable isolation of cells based on SMARCC2 expression levels prior to single-cell genomic analyses

  • This allows correlation between SMARCC2 levels and chromatin accessibility or gene expression patterns at single-cell resolution

  • This approach is particularly valuable for studying heterogeneous tissues or differentiation processes where SMARCC2 expression varies between cells

These emerging applications leverage the fluorescent properties of FITC-conjugated SMARCC2 antibodies to provide unprecedented insights into the dynamic behavior of BAF complexes in chromatin regulation.