SMARCD1 Antibody, FITC conjugated

What is SMARCD1 and what is its functional significance in chromatin biology?

SMARCD1 is a 58 kDa protein that belongs to the nuclear SWI/SNF chromatin remodeling complex. This essential complex plays crucial roles in transcriptional regulation, DNA replication, recombination, and repair processes. As an integral subunit of the SWI/SNF family, SMARCD1 contributes to gene regulation and cellular differentiation. Research has demonstrated that suppression of SWI/SNF components, including SMARCD1, may potentially promote cell proliferation and tumorigenicity . SMARCD1 has particular relevance in brain tissue, with positive detection in both mouse and human brain samples, suggesting important neurological functions .

What applications can SMARCD1 antibodies be used for in research environments?

SMARCD1 antibodies have demonstrated utility across multiple experimental applications:

Each application requires specific optimization for individual experimental conditions .

How does FITC conjugation enhance SMARCD1 antibody functionality in fluorescence-based techniques?

FITC (Fluorescein Isothiocyanate) conjugation to SMARCD1 antibodies provides direct fluorescent visualization without requiring secondary antibody incubation steps. This conjugation offers several advantages for immunofluorescence and flow cytometry applications:

• Reduced protocol time by eliminating secondary antibody incubation

• Decreased background by minimizing non-specific secondary antibody binding

• Compatibility with multicolor staining strategies due to FITC's excitation/emission profile (495nm/519nm)

• Enhanced signal localization precision due to direct antibody labeling

When using FITC-conjugated SMARCD1 antibodies, researchers should be aware that the fluorophore is sensitive to photobleaching and pH changes, requiring appropriate storage conditions and imaging protocols .

What is the subcellular localization pattern of SMARCD1 when detected with fluorescent antibodies?

SMARCD1 predominantly exhibits nuclear localization, consistent with its function in chromatin remodeling complexes. Immunofluorescence experiments using SMARCD1 antibodies in neuroblastoma SH-SY5Y cells reveal distinct nuclear staining patterns with minimal cytoplasmic signal . This nuclear localization pattern correlates with SMARCD1's role in the SWI/SNF chromatin remodeling complex. In glioblastoma research, fluorescent visualization of SMARCD1 has been instrumental in demonstrating altered expression patterns between normal brain tissue and tumor samples, with reduced nuclear SMARCD1 staining intensity observed in high-grade gliomas .

What are the recommended storage and handling conditions for FITC-conjugated SMARCD1 antibodies?

To maintain optimal performance of FITC-conjugated SMARCD1 antibodies:

• Store at -20°C in the dark to prevent photobleaching

• For long-term stability, store in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Antibody aliquoting is generally unnecessary for -20°C storage

• The conjugated antibody remains stable for at least one year after shipment when properly stored

• Avoid repeated freeze-thaw cycles that may compromise antibody performance

• Protect from prolonged light exposure during experimental procedures to prevent fluorophore photobleaching

How does SMARCD1 expression correlate with malignancy in glioblastoma research models?

Studies have revealed a significant inverse correlation between SMARCD1 expression and glioblastoma malignancy. Low expression of SMARCD1 was consistently observed in glioblastoma cell lines (U87 and U251) compared to normal brain tissue. Furthermore, patients with high-grade gliomas demonstrated reduced SMARCD1 expression levels compared to lower-grade tumors .

This relationship has been experimentally validated through both knockdown and overexpression studies:

• SMARCD1 depletion significantly enhanced:

  • Cellular proliferation rates in CCK-8 assays

  • Colony formation capabilities

  • Invasion and migration potential

  • Chemoresistance to temozolomide (TMZ)

• SMARCD1 overexpression conversely resulted in:

  • Inhibited cell proliferation

  • Reduced colony formation

  • Decreased invasion and migration

  • Enhanced sensitivity to chemotherapeutic agents

These findings strongly suggest SMARCD1 functions as a tumor suppressor in glioblastoma, with its decreased expression potentially serving as a biomarker for increased malignancy and poorer prognosis.

What molecular mechanisms underlie SMARCD1's inhibitory effects on glioblastoma cell proliferation?

SMARCD1 exerts its anti-proliferative effects through multiple interconnected mechanisms:

• Cell Cycle Regulation: SMARCD1 overexpression induces G1 phase arrest in glioblastoma cells. Flow cytometry analysis demonstrated significantly increased G1 phase populations in both U87 and U251 cells overexpressing SMARCD1. This correlates with decreased expression of cell cycle regulators CDK4 and cyclin D1, which are essential for G1/S phase transition .

• p53 Pathway Activation: SMARCD1 directly binds to the p53 tumor suppressor protein, as confirmed by immunoprecipitation studies. This interaction enhances p53 stability and promotes activation of downstream targets including p21 (cell cycle inhibitor) and Bax (pro-apoptotic protein). The SMARCD1-p53 axis is particularly important in mediating chemosensitivity to temozolomide .

• Notch1 Pathway Suppression: SMARCD1 negatively regulates the Notch1 signaling pathway, with overexpression of SMARCD1 suppressing Notch1, Hes1, and Hey1 at both protein and mRNA levels. This regulatory mechanism appears independent of BRG1 (the catalytic subunit of the SWI/SNF complex) .

These mechanistic insights highlight SMARCD1's multifaceted role in tumor suppression and suggest potential therapeutic strategies targeting these pathways.

What is the significance of the SMARCD1-Notch1 feedback loop in glioblastoma progression?

Research has uncovered a critical bidirectional regulatory relationship between SMARCD1 and the Notch1 signaling pathway in glioblastoma cells. This regulatory loop appears to be a key determinant of tumor malignancy:

• SMARCD1 regulation of Notch1: Overexpression of SMARCD1 significantly decreases Notch1, Hes1, and Hey1 expression at both protein and mRNA levels. Conversely, SMARCD1 knockdown increases expression of these Notch pathway components .

• Notch1 regulation of SMARCD1: Inhibition of Notch1 signaling through either siRNA or the γ-secretase inhibitor DAPT results in increased SMARCD1 expression. This creates a negative feedback loop where Notch1 represses SMARCD1 expression .

• Hes1 as a mediator: Bioinformatic analysis of the SMARCD1 promoter region identified specific binding sites for Hes1 (a Notch1 target gene). siRNA experiments confirmed that Hes1 knockdown leads to increased SMARCD1 expression in a dose-dependent manner, establishing Hes1 as a transcriptional repressor of SMARCD1 .

This feedback loop has significant implications for understanding glioblastoma progression and potential therapeutic interventions. Breaking this loop through Notch1 inhibition could potentially restore SMARCD1 expression and its tumor-suppressive functions.

How can FITC-conjugated SMARCD1 antibodies be used to investigate cell cycle regulation in cancer models?

FITC-conjugated SMARCD1 antibodies provide valuable tools for investigating SMARCD1's role in cell cycle regulation through several methodological approaches:

• Flow Cytometry Analysis: Dual staining with FITC-conjugated SMARCD1 antibodies and propidium iodide allows simultaneous assessment of SMARCD1 expression levels and cell cycle phase distribution. This approach revealed that SMARCD1 overexpression significantly increased the G1 phase cell population in glioblastoma cells .

• Immunofluorescence Co-localization: FITC-conjugated SMARCD1 antibodies can be paired with antibodies against cell cycle regulators (e.g., CDK4, cyclin D1) using complementary fluorophores. This enables visualization of spatial relationships between SMARCD1 and these regulators during different cell cycle phases.

• Live Cell Imaging: Using FITC-conjugated SMARCD1 antibodies on permeabilized live cells allows for temporal tracking of SMARCD1 dynamics throughout the cell cycle.

• Quantitative Analysis: Digital image analysis of FITC signal intensity provides quantitative measurement of SMARCD1 expression levels in relation to cell cycle phases.

These applications have demonstrated that SMARCD1 plays a crucial role in G1 phase regulation by modulating key cell cycle proteins including CDK4 and cyclin D1 .

What experimental approaches can elucidate SMARCD1's interaction with p53 in chemoresistance mechanisms?

Several experimental strategies utilizing SMARCD1 antibodies have been employed to investigate the SMARCD1-p53 interaction in chemoresistance:

• Co-immunoprecipitation (Co-IP): Studies have demonstrated direct binding between SMARCD1 and p53 through Co-IP experiments. When SMARCD1 was knocked down, decreased p53 binding was observed in the immunoprecipitation pulldown products. Conversely, SMARCD1 overexpression enhanced p53 binding .

• Western Blot Analysis: Following temozolomide (TMZ) treatment, western blotting revealed that SMARCD1 overexpression increased expression of p53 and its downstream targets (p21, Bax) while decreasing anti-apoptotic Bcl-xl. This correlated with increased cleaved Caspase3, indicating enhanced apoptotic activity .

• Apoptosis Assays: Flow cytometry using Annexin V/PI staining showed that SMARCD1 knockdown significantly reduced TMZ-induced apoptosis, while SMARCD1 overexpression sensitized cells to TMZ .

• Chromatin Immunoprecipitation (ChIP): While not explicitly mentioned in the search results, ChIP using SMARCD1 antibodies can identify genomic regions where SMARCD1-containing SWI/SNF complexes regulate p53-target genes.

These approaches collectively demonstrate that SMARCD1 enhances chemosensitivity by directly binding to p53 and activating downstream apoptotic pathways, suggesting potential therapeutic strategies to overcome chemoresistance in glioblastoma.

What are the optimal protocols for immunofluorescence using FITC-conjugated SMARCD1 antibodies?

For optimal immunofluorescence results with FITC-conjugated SMARCD1 antibodies:

• Sample Preparation:

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

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

  • Block with 5% normal serum in PBS for 1 hour

• Antibody Incubation:

  • Dilute FITC-conjugated SMARCD1 antibody 1:20-1:200 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber protected from light

  • Wash 3 times with PBS, 5 minutes each

• Nuclear Counterstaining:

  • Counterstain with DAPI (1:1000) for 5 minutes

  • Wash 3 times with PBS

• Mounting and Imaging:

  • Mount with anti-fade mounting medium

  • Store slides at 4°C in the dark

  • Image using appropriate filter sets (FITC: excitation ~495nm, emission ~519nm)

• Special Considerations:

  • For tissue sections, antigen retrieval may be necessary (suggested: TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Positive controls should include tissues known to express SMARCD1 (e.g., brain tissue)

  • Negative controls should omit primary antibody

This protocol has been validated for detecting SMARCD1 in neuroblastoma SH-SY5Y cells, showing primarily nuclear localization .

What are the key considerations for Western blot analysis using SMARCD1 antibodies?

For reliable Western blot detection of SMARCD1:

• Sample Preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Determine protein concentration using Bradford or BCA assay

  • Load 20-50 μg of total protein per lane

• Electrophoresis and Transfer:

  • Use 10% SDS-PAGE gels for optimal resolution of the 58 kDa SMARCD1 protein

  • Transfer to PVDF membrane (recommended over nitrocellulose for better protein retention)

• Antibody Incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour

  • Dilute SMARCD1 antibody 1:500-1:1000 in blocking solution

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3 times with TBST, 10 minutes each

  • Incubate with HRP-conjugated secondary antibody for 1 hour

  • Wash 3 times with TBST

• Detection and Analysis:

  • Use ECL substrate for visualization

  • Expected molecular weight: 58 kDa

  • Recommended loading controls: GAPDH, β-actin, or β-tubulin

• Validated Positive Controls:

  • Mouse brain tissue

  • Human brain tissue

  • Jurkat cells

When analyzing SMARCD1 in glioblastoma research, researchers should be prepared for potentially weak signals in high-grade glioma samples due to decreased expression levels compared to normal brain tissue .

How should immunoprecipitation experiments be designed to study SMARCD1 interactions?

Immunoprecipitation (IP) is a powerful technique for studying SMARCD1's protein-protein interactions, particularly with p53:

• Lysate Preparation:

  • Use 1.0-3.0 mg of total protein lysate per IP reaction

  • Extract proteins using non-denaturing lysis buffer (e.g., 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA) with protease inhibitors

  • Pre-clear lysate with protein A/G beads to reduce non-specific binding

• Antibody Binding:

  • Use 0.5-4.0 μg of SMARCD1 antibody per IP reaction

  • Incubate with lysate overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate 1-2 hours at 4°C

• Washing and Elution:

  • Wash beads 4-5 times with cold lysis buffer

  • Elute proteins by boiling in SDS sample buffer

• Analysis:

  • Analyze by SDS-PAGE followed by western blotting

  • For SMARCD1-p53 interaction studies, probe with anti-p53 antibody

  • For confirmation of SMARCD1 pulldown, probe with SMARCD1 antibody

• Controls:

  • Input: 5-10% of pre-IP lysate

  • IgG control: Normal rabbit IgG IP to assess non-specific binding

  • Negative control: Lysate from SMARCD1-knockout cells

This approach has been successfully used to demonstrate direct binding between SMARCD1 and p53, with the interaction strength correlating with SMARCD1 expression levels .

What are the critical experimental controls when using FITC-conjugated SMARCD1 antibodies?

When working with FITC-conjugated SMARCD1 antibodies, the following controls are essential:

• Positive Controls:

  • Samples known to express SMARCD1 (e.g., mouse brain tissue, human brain tissue, Jurkat cells)

  • Cell lines with confirmed SMARCD1 expression (e.g., SH-SY5Y cells for immunofluorescence)

• Negative Controls:

  • Isotype control: FITC-conjugated non-specific IgG from the same species

  • SMARCD1 knockout/knockdown cells: Validate antibody specificity

  • Secondary antibody-only control (for unconjugated primary antibodies)

• Signal Validation Controls:

  • Peptide competition: Pre-incubation of antibody with blocking peptide

  • Concentration gradient: Serial dilutions to determine optimal antibody concentration

• Technical Controls for Flow Cytometry:

  • Unstained cells: For autofluorescence assessment

  • Single-color controls: For compensation when performing multicolor analysis

  • Fluorescence minus one (FMO) controls: To determine gating boundaries

• Fluorophore-Specific Controls:

  • Photobleaching control: Repeated imaging of the same field to assess signal stability

  • pH sensitivity control: Testing FITC signal under various buffer conditions

Implementing these controls ensures reliable and interpretable results when using FITC-conjugated SMARCD1 antibodies across different experimental applications.

How can SMARCD1 expression be quantified in immunohistochemistry studies?

Accurate quantification of SMARCD1 expression in immunohistochemistry requires systematic approaches:

• Sample Preparation Optimization:

  • For SMARCD1 IHC, antigen retrieval is critical: use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

  • Standardize section thickness (4-5 μm recommended)

  • Include positive controls (lymphoma tissue has been validated)

• Staining Protocol:

  • Optimal antibody dilution: 1:20-1:200

  • Incubation time: 1-2 hours at room temperature or overnight at 4°C

  • Detection system: DAB chromogen provides good contrast for quantification

• Quantification Methods:

  • H-score method: Calculate H = Σ(Pi × i), where Pi is the percentage of cells with intensity i (0-3)

  • Allred score: Combines proportion score (0-5) and intensity score (0-3)

  • Digital image analysis: Use software like ImageJ with color deconvolution to separate DAB signal

• Standardization Approaches:

  • Use tissue microarrays when possible to ensure uniform staining conditions

  • Include reference slides with known SMARCD1 expression levels

  • Blind scoring by multiple observers to reduce bias

• Result Interpretation:

  • For glioblastoma studies, compare SMARCD1 expression between tumor grades

  • Correlate with clinical parameters (survival, treatment response)

  • Consider subcellular localization (primarily nuclear for SMARCD1)

In glioblastoma research, this approach has revealed significant correlations between reduced SMARCD1 expression and higher tumor grade, providing important prognostic insights .

What are common technical issues when using FITC-conjugated SMARCD1 antibodies and how can they be resolved?

When working with FITC-conjugated SMARCD1 antibodies, researchers may encounter several technical challenges:

• Photobleaching:

  • Problem: Rapid signal fading during microscopy

  • Solution: Use anti-fade mounting media, minimize exposure time, reduce excitation intensity, consider alternative fluorophores for extended imaging sessions

• Background Fluorescence:

  • Problem: High non-specific signal reducing signal-to-noise ratio

  • Solution: Optimize blocking (try 5% BSA or normal serum), increase washing steps, reduce antibody concentration, use proper negative controls

• Weak Signal:

  • Problem: Insufficient SMARCD1 detection

  • Solution: Optimize fixation methods, improve antigen retrieval (try TE buffer pH 9.0), increase antibody concentration, extend incubation time, enhance detection with amplification systems

• Spectral Overlap in Multicolor Experiments:

  • Problem: Bleed-through between channels

  • Solution: Use proper filter sets, perform single-color controls, apply spectral unmixing algorithms during analysis

• Fixation-Related Issues:

  • Problem: Over-fixation masking epitopes or under-fixation causing poor morphology

  • Solution: Test multiple fixation durations, consider alternative fixatives beyond paraformaldehyde

For researchers studying SMARCD1 in glioblastoma models, these optimizations are particularly important given the potentially reduced expression levels in high-grade tumors .

How can contradictory results in SMARCD1 expression studies be reconciled?

Researchers may encounter apparently contradictory findings when studying SMARCD1 expression:

• Tissue-Specific Expression Patterns:

  • Observation: SMARCD1 expression varies significantly between tissue types

  • Reconciliation: Contextualize findings within specific tissue types; brain-derived tissues show different baseline expression than other tissues

• Antibody Specificity Issues:

  • Observation: Different antibodies yield inconsistent results

  • Reconciliation: Validate antibodies using knockdown/knockout controls, compare epitope locations, use multiple antibodies targeting different regions

• Methodology Discrepancies:

  • Observation: Expression varies between protein (Western/IHC) and mRNA (qPCR) levels

  • Reconciliation: Consider post-transcriptional regulation, measure both protein and mRNA when possible, standardize protocols

• SMARCD1-Notch1 Feedback Loop Complexities:

  • Observation: Temporal fluctuations in SMARCD1 expression based on Notch1 activity

  • Reconciliation: Account for the negative feedback loop where Notch1 activation suppresses SMARCD1 expression via Hes1, potentially causing oscillating expression patterns

• Cell Type Heterogeneity:

  • Observation: Variable expression within seemingly homogeneous samples

  • Reconciliation: Use single-cell approaches, microdissection techniques, or cell sorting to isolate specific populations

Understanding these factors has been crucial in resolving apparent contradictions in glioblastoma studies, where SMARCD1's tumor-suppressive role must be interpreted within complex regulatory networks .

What approaches can resolve non-specific binding issues with SMARCD1 antibodies?

Non-specific binding can significantly impact experimental results when using SMARCD1 antibodies:

• Systematic Optimization Strategy:

  • Start with manufacturer's recommended protocol

  • Test a dilution series (broader than the suggested 1:20-1:200 range)

  • Compare multiple blocking agents (BSA, normal serum, commercial blockers)

  • Evaluate different incubation temperatures and durations

• Specific Techniques for Reducing Background:

  • Pre-adsorption: Incubate antibody with proteins from non-target species

  • Increase wash stringency: Higher salt concentration, longer durations, more wash cycles

  • Use highly purified antibody preparations (antigen affinity-purified antibodies are preferred)

• Validation Approaches:

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide

  • SMARCD1 knockdown controls: Confirm signal reduction correlates with expression level

  • Multiple antibodies: Use antibodies targeting different SMARCD1 epitopes

• Application-Specific Solutions:

  • Western Blot: Increase blocking time, optimize transfer conditions

  • Immunofluorescence: Use detergents in wash buffers, test alternative fixation methods

  • Flow Cytometry: Implement proper gating strategies, use viability dyes to exclude dead cells

• Analysis Strategies:

  • Always include isotype controls for baseline subtraction

  • Use digital image analysis to quantify specific vs. non-specific signal

  • Document optimization steps thoroughly for reproducibility

Implementing these approaches ensures more reliable detection of SMARCD1, particularly in contexts where expression levels may be naturally low, such as in high-grade glioblastoma samples .

How should flow cytometry data for FITC-conjugated SMARCD1 antibodies be analyzed?

Flow cytometry with FITC-conjugated SMARCD1 antibodies requires specific analytical approaches:

This analytical framework has successfully demonstrated SMARCD1's role in cell cycle regulation in glioblastoma, particularly its induction of G1 phase arrest when overexpressed .

What strategies can address experimental variability in SMARCD1 research?

Experimental variability in SMARCD1 research can be managed through systematic approaches:

• Sample Preparation Standardization:

  • Standardize cell culture conditions (passage number, confluence, media composition)

  • For patient-derived samples, document clinical parameters and processing timeframes

  • Use consistent lysate preparation protocols with standardized buffer compositions

• Technical Replication Strategy:

  • Perform both technical replicates (same sample, multiple measurements) and biological replicates (independent samples)

  • For Western blot, load multiple concentrations to ensure signal linearity

  • In immunofluorescence, image multiple fields across different samples

• Internal Controls Implementation:

  • Include reference cell lines with known SMARCD1 expression levels

  • Use housekeeping proteins consistently across experiments

  • Implement spike-in controls where appropriate

• Data Normalization Methods:

  • For Western blot: Normalize to loading controls (GAPDH, β-actin)

  • For IHC/IF: Use background subtraction and reference intensity standards

  • For qPCR: Select stable reference genes validated for your experimental system

• Statistical Analysis Approach:

  • Determine appropriate sample sizes through power analysis

  • Use statistical methods that account for experimental design (e.g., mixed models for repeated measures)

  • Report effect sizes alongside p-values

  • Document outlier criteria and handling

These approaches have been essential in establishing reliable findings regarding SMARCD1's tumor-suppressive role in glioblastoma, particularly in studies analyzing the complex regulatory relationship with the Notch1 pathway .