SMARCD2 Antibody, FITC conjugated

What is SMARCD2 and what cellular functions does it perform?

SMARCD2, also known as BAF60B, is a subunit of the SWI/SNF chromatin remodeling complex that plays a crucial role in controlling gene expression and cell fate determination. It functions primarily in the nucleus where it participates in chromatin remodeling to regulate accessibility of transcription factors to DNA. SMARCD2 has been directly implicated in transcriptional regulation through its ability to remodel chromatin structure, particularly in processes related to hematopoietic differentiation. Loss-of-function mutations in SMARCD2 have been associated with immunodeficiency and developmental disorders, suggesting its essential role in normal cellular development and function .

What are the key characteristics of FITC-conjugated SMARCD2 antibodies?

FITC-conjugated SMARCD2 antibodies typically consist of rabbit polyclonal antibodies specific to human SMARCD2 protein that have been chemically linked to fluorescein isothiocyanate (FITC). These antibodies are generated using recombinant fusion proteins of human SMARCD2 as immunogens, often targeting specific amino acid sequences. The FITC fluorophore has excitation/emission wavelengths of approximately 495/519 nm, making it compatible with the 488 nm laser line commonly used in flow cytometry and fluorescence microscopy . The antibodies typically recognize the full-length SMARCD2 protein with a calculated molecular weight of approximately 59-64 kDa and are designed to maintain high specificity while exhibiting minimal background fluorescence .

How do FITC-conjugated antibodies differ from other fluorophore conjugates for SMARCD2 detection?

FITC-conjugated SMARCD2 antibodies provide distinct advantages and limitations compared to other fluorophore conjugates. FITC emits green fluorescence (emission peak ~519 nm) which contrasts with other available conjugates such as AF350 (emission ~442 nm), AF405 (emission ~421 nm), AF555 (emission ~565 nm), and AF647 (emission ~667 nm) . This spectral characteristic allows for multicolor experimental design, but researchers must consider that FITC has relatively rapid photobleaching compared to Alexa Fluor conjugates. Additionally, FITC fluorescence is pH-sensitive and optimal at slightly alkaline conditions (pH >7.5), whereas other conjugates like AF594 or AF647 maintain stable fluorescence across a broader pH range. In multiplexed experiments, FITC can be effectively combined with red-emitting fluorophores (e.g., AF647) while minimizing spectral overlap, though careful compensation controls are required when used with PE (phycoerythrin) due to potential bleed-through .

What is the optimal protocol for immunofluorescence staining using FITC-conjugated SMARCD2 antibody?

For optimal immunofluorescence staining with FITC-conjugated SMARCD2 antibody, follow this methodological approach:

• Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes (critical for nuclear antigen access)

• Blocking and Antibody Incubation:

  • Block with 5% BSA in PBS-T (PBS with 0.1% Tween-20) for 1 hour at room temperature

  • Incubate with FITC-conjugated SMARCD2 antibody (typically 1:50-1:200 dilution) in blocking buffer for 2 hours at room temperature or overnight at 4°C in a humidified dark chamber

• Nuclear Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium specifically formulated for fluorescence preservation

• Imaging Parameters:

  • Use appropriate filter sets (excitation: 495±10 nm, emission: 519±10 nm)

  • Minimize exposure time to prevent photobleaching of FITC

  • Acquire Z-stack images to fully capture nuclear localization of SMARCD2

For optimal results, maintain samples in darkness whenever possible and process all experimental and control samples identically. Include appropriate controls including secondary antibody-only controls to assess background fluorescence .

How should researchers validate the specificity of FITC-conjugated SMARCD2 antibodies in their experimental system?

Validating the specificity of FITC-conjugated SMARCD2 antibodies requires a multi-faceted approach:

• Positive and Negative Control Tissues/Cells:

  • Positive controls: Mouse and rat thymus tissues, which are known to express SMARCD2

  • Negative controls: Tissues/cell lines with confirmed SMARCD2 knockdown or knockout

• Competitive Peptide Blocking:

  • Pre-incubate the antibody with excess immunizing peptide (sequence corresponding to amino acids 454-531 of human SMARCD2) prior to staining

  • A significant reduction in signal indicates specificity for the target epitope

• Multi-antibody Validation:

  • Compare staining patterns using different antibodies targeting distinct SMARCD2 epitopes

  • Concordant staining patterns support specificity

• Genetic Validation:

  • Perform parallel experiments with SMARCD2 knockdown/knockout cells

  • Reduced or absent signal in these samples confirms specificity

  • Use siRNA or CRISPR-Cas9 systems targeting SMARCD2

• Western Blot Correlation:

  • Perform Western blot analysis alongside immunofluorescence

  • Confirm the antibody detects a single band at the expected molecular weight (59-64 kDa)

• Cross-reactivity Assessment:

  • Test the antibody on samples from different species to confirm the stated species reactivity (Human, Mouse, Rat)

  • Evaluate potential cross-reactivity with other SMARCD family members (SMARCD1, SMARCD3)

Documentation of these validation steps is essential for publication-quality research and reproducibility .

What are the recommended sample preparation methods for flow cytometry using FITC-conjugated SMARCD2 antibodies?

For flow cytometry analysis using FITC-conjugated SMARCD2 antibodies, follow these methodological guidelines:

• Cell Preparation:

  • Harvest cells in exponential growth phase (1-5 × 10^6 cells per sample)

  • Wash twice with ice-cold PBS containing 2% FBS

  • Maintain samples at 4°C throughout preparation to preserve cellular integrity

• Fixation and Permeabilization (Critical for Nuclear Antigen):

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with either:
    a) 0.1% Triton X-100 in PBS for 10 minutes at room temperature, or
    b) 90% ice-cold methanol for 30 minutes on ice (preferred for robust nuclear permeabilization)

• Blocking and Antibody Staining:

  • Block with 5% normal serum in PBS for 30 minutes at room temperature

  • Incubate with FITC-conjugated SMARCD2 antibody (typically 1:50-1:100 dilution) for 45-60 minutes at room temperature in the dark

  • Wash three times with PBS containing 2% FBS

• Controls and Analysis Parameters:

  • Include unstained cells, isotype control, and single-color controls for compensation

  • Analyze using 488 nm laser excitation and 525/40 nm bandpass filter

  • Set PMT voltage to position negative population in first decade of log scale

  • Collect minimum of 10,000 events per sample

• Data Analysis Considerations:

  • Gate on single cells using FSC-H vs. FSC-A to exclude doublets

  • Gate on viable cells if using a viability dye

  • Present data as histogram overlays or median fluorescence intensity values

For optimal results, prepare freshly conjugated antibody or use within 6 months when stored protected from light at 4°C .

How can FITC-conjugated SMARCD2 antibodies be used to investigate SWI/SNF complex assembly and function?

FITC-conjugated SMARCD2 antibodies can be strategically employed to investigate SWI/SNF complex assembly and function through several advanced methodological approaches:

• Co-immunoprecipitation with Fluorescence Detection:

  • Use FITC-conjugated SMARCD2 antibodies to immunoprecipitate the protein complex

  • Analyze co-precipitating partners such as SMARCA4 (BRG1), SMARCC2 (BAF170), SMARCC1 (BAF155), and SMARCB1 (BAF47)

  • Quantify relative fluorescence intensity to assess complex composition under different cellular conditions

• Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq):

  • Implement a modified ChIP protocol using FITC-conjugated SMARCD2 antibodies

  • Isolate SMARCD2-bound chromatin regions

  • Sequence and analyze to identify genomic binding sites and assess overlap with transcription factor binding sites

  • Compare binding profiles in wild-type versus mutant cells to understand functional implications

• FRET (Förster Resonance Energy Transfer) Analysis:

  • Combine FITC-conjugated SMARCD2 antibodies with another SWI/SNF component antibody labeled with a compatible acceptor fluorophore

  • Measure FRET efficiency to assess protein-protein proximity within the complex

  • Apply in live or fixed cells to study dynamic changes in complex assembly

• High-Content Screening with Quantitative Image Analysis:

  • Utilize automated microscopy platforms to analyze SMARCD2 localization across thousands of cells

  • Implement machine learning algorithms to classify subcellular distribution patterns

  • Correlate with functional readouts during differentiation or disease progression

This multimodal approach enables researchers to decipher both compositional and functional aspects of SWI/SNF complexes containing SMARCD2, providing insights into chromatin remodeling mechanisms that regulate gene expression networks critical for cellular differentiation and disease pathogenesis .

What experimental design considerations should be made when studying SMARCD2 mutations using fluorescence-based techniques?

When studying SMARCD2 mutations using fluorescence-based techniques, researchers should implement the following experimental design considerations:

• Mutation-Specific Epitope Accessibility Assessment:

  • Determine whether the FITC-conjugated antibody's epitope region (e.g., amino acids 454-531) is affected by the mutation being studied

  • For frameshift mutations like p.Arg73Valfs*8, assess potential truncation effects on epitope recognition

  • Consider multiple antibodies targeting different epitopes for comprehensive analysis

• Expression Vector System Design:

  • Develop FLAG-tagged expression vectors carrying both wild-type and mutated SMARCD2 versions

  • Include an IRES-RFP (Internal Ribosomal Entry Sequence-Red Fluorescence Protein) for transfection efficiency monitoring

  • Create stable cell lines for long-term studies of mutation effects

• Quantitative Co-localization Analysis:

  • Implement rigorous co-localization metrics (Pearson's correlation coefficient, Manders' overlap coefficient)

  • Compare wild-type and mutant SMARCD2 co-localization with other SWI/SNF components

  • Use high-resolution techniques (confocal, super-resolution microscopy) for precise spatial information

• Functional Rescue Experimental Design:

  • Design complementation studies with fluorescence readouts

  • Express wild-type SMARCD2 in mutant/knockout backgrounds

  • Quantify restoration of proper nuclear localization and complex formation

• Controls for Fluorescence Studies:

  • Include both positive controls (cells known to express SMARCD2) and negative controls (SMARCD2-knockout cells)

  • Use mouse and rat thymus as positive control tissues

  • Account for potential autofluorescence in tissues with high collagen/elastin content

• Multiparametric Flow Cytometry Panel Design:

  • Combine FITC-conjugated SMARCD2 antibody with markers of cell differentiation

  • Implement careful compensation when using FITC alongside PE-conjugated antibodies

  • Include appropriate FMO (Fluorescence Minus One) controls

This comprehensive approach enables accurate assessment of how SMARCD2 mutations affect protein localization, complex formation, and function in various cellular contexts .

How can researchers implement multiplexed imaging with FITC-conjugated SMARCD2 antibodies alongside other fluorophores?

Implementing multiplexed imaging with FITC-conjugated SMARCD2 antibodies requires careful optimization of several technical parameters:

• Strategic Fluorophore Selection:

  • Pair FITC (excitation 495 nm/emission 519 nm) with spectrally distinct fluorophores

  • Optimal companions include:

    • AF594 (excitation 591 nm/emission 614 nm)

    • AF647 (excitation 651 nm/emission 667 nm)

    • AF405 (excitation 401 nm/emission 421 nm)

  • Avoid PE conjugates when possible due to spectral overlap with FITC

• Sequential Staining Protocol:

  • Block with 5% BSA in PBS-T for 1 hour at room temperature

  • Apply primary antibodies sequentially for multi-species antibodies

  • For nuclear SMARCD2 detection, apply the FITC-conjugated antibody last in the sequence

  • Include 10-minute PBS washes (3×) between each antibody application

• Image Acquisition Parameters:

  • Capture single-color controls for spectral unmixing

  • Implement sequential scanning rather than simultaneous acquisition

  • Begin with FITC channel (most susceptible to photobleaching)

  • Optimize exposure settings individually for each channel

• Post-Acquisition Analysis:

  • Apply spectral unmixing algorithms to remove bleed-through

  • Implement colocalization analysis using Pearson's or Manders' coefficients

  • Utilize 3D reconstruction for nuclear proteins like SMARCD2

• Experimental Controls for Multiplexed Imaging:

  • Single-label controls for each fluorophore

  • FMO (Fluorescence Minus One) controls

  • Secondary antibody-only controls to assess background

  • Absorption controls to ensure antibody penetration into nuclear regions

This comprehensive approach enables simultaneous visualization of SMARCD2 alongside other proteins of interest, allowing researchers to investigate protein interactions within the SWI/SNF complex and its relationship with other nuclear components .

What are common issues encountered with FITC-conjugated antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with FITC-conjugated SMARCD2 antibodies. Here are the most common issues and their methodological solutions:

• Photobleaching:

  • Problem: Rapid signal loss during imaging due to FITC's susceptibility to photobleaching

  • Solutions:

    • Add anti-fade agents (e.g., ProLong Gold, SlowFade) to mounting media

    • Reduce exposure time and lamp intensity during imaging

    • Acquire FITC channel first in multiplexed experiments

    • Consider switching to more photostable AF488 conjugates for extended imaging sessions

• pH Sensitivity:

  • Problem: Decreased fluorescence in acidic environments

  • Solutions:

    • Maintain buffers at pH 7.4-8.0 throughout all experimental steps

    • Avoid acidic fixatives when possible

    • Use buffered mounting media specifically designed for fluorescence preservation

• High Background Fluorescence:

  • Problem: Poor signal-to-noise ratio

  • Solutions:

    • Implement more stringent blocking (5% BSA, 5% normal serum, 0.3% Triton X-100)

    • Increase wash duration and number (minimum 3 washes, 5 minutes each)

    • Optimize antibody dilution (typically 1:50-1:200)

    • Include 0.1% Sudan Black B in 70% ethanol to reduce autofluorescence in tissues

• Inadequate Nuclear Signal:

  • Problem: Weak or absent nuclear staining despite SMARCD2's nuclear localization

  • Solutions:

    • Ensure robust permeabilization (0.5% Triton X-100 for 15 minutes or methanol fixation)

    • Implement heat-mediated antigen retrieval (citrate buffer pH 6.0 or TE buffer pH 9.0)

    • Extend primary antibody incubation to overnight at 4°C

    • Verify antibody functionality with positive control samples (mouse/rat thymus)

• Spectral Overlap in Multiplexed Experiments:

  • Problem: Bleed-through between FITC and other channels

  • Solutions:

    • Implement rigorous compensation controls

    • Use sequential scanning rather than simultaneous acquisition

    • Apply post-acquisition spectral unmixing algorithms

    • Redesign panel to use more spectrally distinct fluorophores

By systematically addressing these technical challenges, researchers can significantly improve the quality and reliability of their SMARCD2 detection experiments.

How can researchers validate experimental results obtained with FITC-conjugated SMARCD2 antibodies?

To ensure scientific rigor when working with FITC-conjugated SMARCD2 antibodies, researchers should implement the following validation approaches:

• Orthogonal Detection Methods:

  • Confirm fluorescence microscopy findings with independent techniques:

    • Western blot analysis using unconjugated antibodies (expected MW: 59-64 kDa)

    • Immunohistochemistry with enzymatic detection systems

    • Mass spectrometry-based protein identification

  • Compare results across multiple detection platforms to confirm consistency

• Genetic Validation Strategies:

  • Implement SMARCD2 knockdown/knockout controls:

    • siRNA-mediated transient knockdown

    • CRISPR-Cas9 engineered knockout cell lines

    • Conditional knockout models for tissue-specific validation

  • Document proportional signal reduction corresponding to protein depletion level

• Antibody Cross-Validation:

  • Compare results using multiple antibodies targeting different SMARCD2 epitopes

  • Validate with both polyclonal and monoclonal antibodies when available

  • Test antibodies from different vendors with documented epitope information

• Functional Correlation Studies:

  • Link antibody-detected SMARCD2 localization/expression with functional readouts:

    • Chromatin accessibility changes (ATAC-seq)

    • Transcriptional profiling (RNA-seq)

    • Phenotypic assays relevant to SMARCD2 function

• Statistical Robustness Measures:

  • Implement quantitative image analysis:

    • Measure fluorescence intensity across multiple fields/samples

    • Quantify nuclear/cytoplasmic distribution ratios

    • Apply appropriate statistical tests (minimum n=3 biological replicates)

  • Report effect sizes and confidence intervals rather than just p-values

• Reproducibility Assessment:

  • Test antibody performance across:

    • Multiple lots

    • Different experimental days

    • Independent operators

  • Document all experimental conditions thoroughly to enable reproduction

By implementing these rigorous validation strategies, researchers can establish confidence in their experimental results and contribute to reliable knowledge advancement in SMARCD2 biology .

How might FITC-conjugated SMARCD2 antibodies be utilized in single-cell analysis techniques?

FITC-conjugated SMARCD2 antibodies present powerful opportunities for single-cell analysis through several cutting-edge methodological approaches:

• Single-Cell Imaging Flow Cytometry:

  • Combine quantitative flow cytometry with high-resolution imaging

  • Measure SMARCD2 nuclear localization intensity alongside morphological parameters

  • Correlate SMARCD2 expression with cell cycle phases using DNA content staining

  • Implement machine learning algorithms to classify cellular subpopulations based on SMARCD2 distribution patterns

• Mass Cytometry (CyTOF) Integration:

  • Develop metal-tagged SMARCD2 antibodies based on validated FITC-conjugated clones

  • Integrate into high-dimensional panels (30+ parameters) including:

    • Other SWI/SNF complex members

    • Transcription factors

    • Chromatin modification markers

  • Apply dimensionality reduction techniques (tSNE, UMAP) to identify novel cellular states

• Single-Cell Multi-omics Applications:

  • Implement CITE-seq approaches combining:

    • FITC-conjugated SMARCD2 antibody detection

    • Single-cell transcriptomics

    • Chromatin accessibility profiling

  • Correlate protein levels with gene expression and chromatin states at single-cell resolution

• Spatial Transcriptomics Integration:

  • Combine FITC-conjugated SMARCD2 immunofluorescence with in situ transcriptomics

  • Map spatial relationships between SMARCD2-expressing cells and specific transcriptional programs

  • Develop computational approaches to correlate protein localization with spatially resolved gene expression

• Live-Cell Imaging Applications:

  • Utilize cell-permeable nanobody-based derivatives of validated SMARCD2 antibodies

  • Track dynamic changes in SMARCD2 localization during differentiation or disease progression

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess SMARCD2 mobility within nuclear compartments

These advanced single-cell approaches will enable researchers to decipher heterogeneity in SMARCD2 expression and function across diverse cell populations, potentially revealing novel insights into its role in development, differentiation, and disease pathogenesis .

What are emerging research areas where FITC-conjugated SMARCD2 antibodies could provide valuable insights?

Several cutting-edge research areas could benefit substantially from applications of FITC-conjugated SMARCD2 antibodies:

• Hematopoietic Stem Cell Differentiation:

  • Track SMARCD2 expression during neutrophil differentiation stages

  • Investigate correlation between SMARCD2 localization and lineage commitment decisions

  • Develop predictive markers for differentiation potential based on SMARCD2 expression patterns

• Chromatin Architecture and Nuclear Organization:

  • Combine with super-resolution microscopy (STORM, PALM) to map SMARCD2 distribution at nanoscale resolution

  • Investigate co-localization with topologically associating domains (TADs) and chromatin loops

  • Explore potential role in phase separation within the nucleus through quantitative imaging

• Cancer Biology and Precision Medicine:

  • Profile SMARCD2 expression across tumor subtypes using tissue microarrays

  • Correlate with treatment response and patient outcomes

  • Develop potential diagnostic or prognostic applications based on SMARCD2 expression patterns

• Developmental Biology:

  • Map SMARCD2 expression during embryonic development in model organisms

  • Investigate role in tissue-specific differentiation programs

  • Explore potential developmental origins of SMARCD2-associated disorders

• Immunological Research:

  • Investigate SMARCD2's role in immune cell function and differentiation

  • Explore potential connections to immunodeficiency disorders

  • Develop therapeutic strategies targeting SMARCD2-dependent pathways

• Regenerative Medicine:

  • Utilize FITC-conjugated SMARCD2 antibodies to monitor cellular reprogramming

  • Assess SMARCD2 dynamics during induced pluripotent stem cell generation

  • Explore potential manipulation of SMARCD2 to enhance differentiation efficiency

These emerging research areas represent fertile ground for application of FITC-conjugated SMARCD2 antibodies, particularly as they relate to understanding fundamental mechanisms of chromatin remodeling in development and disease contexts .

What storage and handling recommendations ensure optimal performance of FITC-conjugated SMARCD2 antibodies?

Maintaining optimal performance of FITC-conjugated SMARCD2 antibodies requires adherence to specific storage and handling protocols:

Storage Conditions:

• Store at 4°C in the dark for up to 6 months

• For long-term storage, aliquot into single-use volumes and store at -20°C

• Avoid repeated freeze-thaw cycles (limit to maximum 3 cycles)

• Protect from light at all times using amber tubes or aluminum foil wrapping

Buffer Composition:

• Optimal buffer: 0.01M Sodium Phosphate, 0.25M NaCl, pH 7.6, with 5mg/ml Bovine Serum Albumin and 0.02% Sodium Azide

• This formulation maintains antibody stability while preventing microbial contamination

Working Solution Preparation:

• Thaw aliquots completely before use and mix gently by inversion (avoid vortexing)

• Centrifuge briefly (30 seconds at 10,000g) to collect contents before opening

• Prepare working dilutions immediately before use

• Do not store diluted antibody solutions for extended periods

Quality Control Measures:

• Periodically validate antibody performance using positive control samples (mouse/rat thymus tissue)

• Monitor for signs of degradation:

  • Diminished fluorescence intensity

  • Increased background staining

  • Altered staining pattern

• Implement reference standards to ensure consistent performance across experiments

Handling Precautions:

• Minimize exposure to light during all preparation and experimental steps

• Work under reduced ambient lighting when preparing solutions

• Use low-retention tubes and pipette tips to prevent protein adsorption

• Implement aseptic technique to prevent microbial contamination

By adhering to these storage and handling recommendations, researchers can maximize the lifespan and performance consistency of their FITC-conjugated SMARCD2 antibodies across multiple experiments .

What resources are available for researchers seeking to implement advanced SMARCD2 studies?

Researchers pursuing advanced studies of SMARCD2 can access various specialized resources:

Biological Resources:

• Validated Cell Lines: SH-SY5Y (human neuroblastoma) and Jurkat (T lymphocyte) cell lines express detectable levels of SMARCD2

• Tissue Resources: Mouse and rat thymus tissue serve as reliable positive controls

• Genetic Models: SMARCD2 knockout/knockdown systems available through repositories like Jackson Laboratory and Addgene

Technical Resources:

• Optimized Protocols:

  • ChIP-seq protocols specifically adapted for SWI/SNF complex components

  • Co-immunoprecipitation methods for studying protein-protein interactions within the complex

  • Immunofluorescence procedures for optimal nuclear antigen detection

Bioinformatics Tools:

• Genomic Databases: ChIP-Atlas and ENCODE contain datasets on SMARCD2 binding sites

• Protein Interaction Databases: BioGRID and STRING provide comprehensive interaction networks

• Gene Expression Resources: GTEx and Human Protein Atlas offer tissue-specific expression data

Research Networks and Collaborations:

• Chromatin Remodeling Consortia: International networks focused on SWI/SNF complex biology

• Rare Disease Networks: Resources for researchers studying SMARCD2-related immunodeficiencies

• Technology-Specific Communities: Forums for advanced imaging and single-cell analysis methods

Experimental Design Support:

• Control Recommendations:

  • Positive controls: SMARCD2-expressing tissues (thymus, brain)

  • Negative controls: SMARCD2 knockdown/knockout systems

  • Technical controls: Isotype-matched, FITC-conjugated irrelevant antibodies

Specialized Methodologies:

• Advanced Imaging: Super-resolution microscopy protocols optimized for nuclear proteins

• Flow Cytometry: Multiparameter panels incorporating SMARCD2 detection

• Mass Spectrometry: Protocols for SWI/SNF complex purification and analysis