SMARCD1 Antibody, Biotin conjugated

What is SMARCD1 and what is its role in cellular processes?

SMARCD1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1), also known as BAF60a, is a 476 amino acid protein that localizes to the nucleus and contains one SWIB domain. It functions as a component of the BAF complex and plays an essential role in chromatin remodeling. SMARCD1 influences vitamin D-mediated transcriptional activity and provides a link between the vitamin D receptor (VDR) and SWI/SNF chromatin remodeling complexes. It is expressed ubiquitously with notable expression in liver, brain, muscle, lung, kidney, pancreas, and placenta . Recent research demonstrates that SMARCD1 negatively regulates myeloid differentiation of leukemic cells, highlighting its potential role in maintaining the undifferentiated state of leukemic cells .

What applications are biotin-conjugated SMARCD1 antibodies validated for?

Based on product information, biotin-conjugated SMARCD1 antibodies (such as A64339) are primarily validated for ELISA applications . The biotin conjugation enhances detection sensitivity through the strong affinity between biotin and streptavidin molecules. The specific biotin-conjugated SMARCD1 antibody described recognizes human SMARCD1 and is developed from rabbit hosts using recombinant Human SMARCD1 protein (amino acids 7-124) as the immunogen . While the product information specifically mentions ELISA validation, biotin-conjugated antibodies generally offer versatility for other detection systems requiring signal amplification.

How do I properly store biotin-conjugated SMARCD1 antibodies to maintain activity?

Biotin-conjugated SMARCD1 antibodies should be stored at -20°C or -80°C upon receipt. It is important to avoid repeated freeze-thaw cycles that could compromise antibody integrity and function . The antibody is typically provided in liquid form with a preservative (0.03% Proclin 300) in a buffer consisting of 50% Glycerol and 0.01M PBS at pH 7.4 . These storage conditions help maintain the structural integrity of both the antibody and the biotin conjugate. Before using the antibody after storage, it is recommended to allow it to equilibrate to room temperature and gently mix before opening to ensure homogeneity.

How should researchers optimize ELISA protocols using biotin-conjugated SMARCD1 antibodies?

When optimizing ELISA protocols with biotin-conjugated SMARCD1 antibodies, researchers should:

• Perform titration experiments to determine the optimal antibody concentration that provides maximal specific signal with minimal background.

• Evaluate different blocking agents (BSA, casein, commercial blockers) to minimize non-specific binding.

• Optimize incubation times and temperatures for both primary antibody and streptavidin-conjugated detection reagents.

• Include proper controls:

  • Positive controls: samples known to express SMARCD1

  • Negative controls: samples lacking SMARCD1 expression

  • Technical controls: wells without primary antibody to assess non-specific binding

• Consider pre-blocking samples with avidin/streptavidin to reduce background from endogenous biotin.

• Optimize washing steps between reagent additions to reduce background while preserving specific signals.

What factors should be considered when selecting between unconjugated and biotin-conjugated SMARCD1 antibodies?

When choosing between unconjugated and biotin-conjugated SMARCD1 antibodies, researchers should consider:

• Application requirements: Biotin-conjugated antibodies provide signal amplification advantages for applications requiring enhanced sensitivity, while unconjugated antibodies offer more flexibility in detection methods.

• Sample type: Tissues or cells with high endogenous biotin (such as liver, kidney) may yield higher background with biotin-conjugated antibodies.

• Detection system availability: Biotin-conjugated antibodies require streptavidin-coupled detection reagents.

• Multiplexing needs: When performing multi-color detection, biotin-conjugated antibodies can be paired with directly-labeled antibodies against other targets.

• Research question: For co-immunoprecipitation studies investigating protein-protein interactions, unconjugated antibodies may be preferable to avoid potential steric hindrance from the biotin moiety .

• Epitope accessibility: The biotin conjugation might affect antibody binding to certain epitopes, particularly in conformationally sensitive applications.

How can I validate the specificity of SMARCD1 antibody signals in my experimental system?

To validate SMARCD1 antibody specificity, implement these approaches:

• Perform knockdown/knockout experiments using siRNA, shRNA, or CRISPR-Cas9 to confirm corresponding reduction in antibody signal. This approach was successfully employed in studies examining SMARCD1's role in myeloid differentiation .

• Compare staining patterns across multiple antibodies targeting different SMARCD1 epitopes to confirm consistent detection patterns.

• Employ peptide competition assays using the immunogen peptide (recombinant Human SMARCD1 protein, amino acids 7-124 for specific antibodies) to block specific binding .

• Compare observed expression patterns with published literature on SMARCD1 expression in different tissues and cell types.

• For biotin-conjugated antibodies specifically, include controls to account for potential background signals from endogenous biotin.

• Verify antibody specificity via Western blot to confirm detection of SMARCD1 at the expected molecular weight (approximately 58 kDa) .

How can SMARCD1 antibodies be used to investigate chromatin remodeling mechanisms?

SMARCD1 antibodies can be powerful tools for investigating chromatin remodeling mechanisms through these approaches:

• Chromatin Immunoprecipitation (ChIP): SMARCD1 antibodies can be used to identify genomic regions where SMARCD1 is recruited. Research has shown that SMARCD1 associates with the SWI/SNF complex and is recruited to promoter regions of myeloid differentiation-specific genes .

• Co-immunoprecipitation (Co-IP): SMARCD1 antibodies can pull down associated proteins to identify interaction partners within the SWI/SNF complex. This approach has confirmed that SMARCD1 associates with SMARCA4, a core component of the SWI/SNF complex .

• Sequential ChIP: By performing sequential immunoprecipitation with antibodies against SMARCD1 and other chromatin remodeling factors, researchers can identify genomic regions where multiple factors co-occupy.

• Immunofluorescence microscopy: Biotin-conjugated SMARCD1 antibodies can be used to visualize the nuclear localization of SMARCD1 and its co-localization with other chromatin-associated factors.

• Proximity ligation assays: These can detect direct interactions between SMARCD1 and other nuclear proteins in situ, providing spatial information about complex formation.

What approaches can researchers use to study SMARCD1's role in myeloid differentiation and leukemia?

Based on recent findings showing SMARCD1's negative regulation of myeloid differentiation in leukemic cells , researchers can:

• Track SMARCD1 expression changes during myeloid differentiation using Western blot, immunofluorescence, or flow cytometry with SMARCD1 antibodies.

• Implement ChIP-qPCR to monitor SMARCD1 recruitment to promoters of myeloid differentiation genes before and after differentiation induction.

• Compare SMARCD1 expression levels across different FAB subtypes of AML, as research shows higher expression in undifferentiated AML subtypes (M0, M1, M2) compared to more differentiated subtypes (M3, M4, M5) .

• Assess SMARCD1 expression in normal versus leukemic stem/progenitor cells to understand its contribution to leukemia maintenance.

• Monitor changes in SMARCD1 levels in response to differentiation-inducing agents such as vitamin D3, which has been shown to reduce SMARCD1 expression in HL-60 cells .

• Investigate how SMARCD1 knockdown enhances sensitivity to differentiation agents, providing insights into potential therapeutic approaches for AML.

How can researchers distinguish between functions of SMARCD1, SMARCD2, and SMARCD3 isoforms in hematopoietic systems?

Research indicates complex interplay between SMARCD family members during hematopoietic differentiation . To differentiate their functions:

• Use isoform-specific antibodies to track expression patterns during differentiation. SMARCD1 and SMARCD2 show high expression in CD34+ hematopoietic stem/progenitor cells, while SMARCD3 is enriched in monocytes .

• Monitor isoform switching during differentiation, as SMARCD1 expression decreases while SMARCD3 increases during myeloid differentiation of cord blood-derived CD34+ cells and HL-60 cells .

• Perform isoform-specific knockdown experiments to assess the impact on differentiation markers and cellular morphology.

• Use ChIP-seq with isoform-specific antibodies to map distinct and overlapping genomic targets.

• Compare the effects of differentiation-inducing agents on expression of each isoform to understand their regulation.

• Implement rescue experiments where one isoform is expressed following knockdown of another to assess functional redundancy.

What controls should be included when using biotin-conjugated SMARCD1 antibodies?

When using biotin-conjugated SMARCD1 antibodies, these controls are essential:

• Isotype control: Use biotin-conjugated rabbit IgG (matching the host species of the SMARCD1 antibody) to assess non-specific binding.

• Blocking control: Pre-incubate the antibody with recombinant SMARCD1 protein (the immunogen) to confirm specificity.

• Endogenous biotin control: Include samples treated only with streptavidin-conjugated detection reagent (no primary antibody) to assess background from endogenous biotin.

• Positive control: Include samples known to express SMARCD1, such as Jurkat cells or brain tissue, which have been confirmed to express SMARCD1 .

• Negative control: Where possible, include SMARCD1 knockdown or knockout samples. Studies have successfully used shRNA to knockdown SMARCD1 in HL-60 and U937 cell lines .

• Processing control: Process some samples without the primary antibody but with all other reagents to identify any non-specific binding from the detection system.

How can researchers address non-specific binding and high background when using SMARCD1 antibodies?

To minimize non-specific binding and high background:

• Optimize blocking conditions by testing different blocking agents (BSA, normal serum, commercial blockers) at various concentrations.

• For biotin-conjugated antibodies, use avidin/streptavidin blocking kits to neutralize endogenous biotin, especially in biotin-rich tissues.

• Increase washing duration and frequency between steps to remove unbound antibody.

• Titrate the antibody concentration to find the optimal dilution that maximizes specific signal while minimizing background.

• Include detergents (0.05-0.1% Tween-20) in wash buffers to reduce non-specific hydrophobic interactions.

• For tissue sections, block endogenous peroxidase activity (for HRP-based detection systems) using hydrogen peroxide treatment.

• Consider using monovalent Fab fragments to block endogenous immunoglobulins in tissue samples.

• Pre-absorb the antibody with tissue homogenates from negative control samples to reduce non-specific interactions.

What are the optimal sample preparation methods for detecting SMARCD1 in different experimental systems?

Optimal sample preparation varies by application and sample type:

• For Western blot:

  • Extract nuclear proteins using specialized nuclear extraction buffers, as SMARCD1 is a nuclear protein.

  • Use protease inhibitors to prevent degradation during extraction.

  • Recommended dilution for Western blot applications is 1:500-1:1000 based on antibody validation data .

• For immunoprecipitation:

  • Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

  • Include gentle detergents in lysis buffers to maintain protein-protein interactions within the SWI/SNF complex.

• For immunohistochemistry:

  • For FFPE tissues, antigen retrieval is crucial - use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 .

  • Recommended dilution for IHC applications is 1:20-1:200 .

• For immunofluorescence:

  • Ensure adequate cell permeabilization to allow antibody access to nuclear SMARCD1.

  • Recommended dilution for IF applications is 1:20-1:200 .

  • Fix cells with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100.

How can researchers interpret conflicting results between different SMARCD1 detection methods?

When encountering conflicting results between different detection methods:

• Consider epitope accessibility differences:

  • Some epitopes may be masked in certain applications (e.g., formalin fixation can mask epitopes in IHC).

  • Certain protein interactions may block antibody binding sites in specific cellular contexts.

• Evaluate protein complex dynamics:

  • SMARCD1 exists in protein complexes, and complex formation may affect antibody binding.

  • Co-immunoprecipitation studies have confirmed SMARCD1's association with the SWI/SNF complex , which may impact detection in different contexts.

• Assess experimental conditions:

  • Different buffers, pH conditions, or detergents used across methods may affect antibody-antigen interactions.

  • Denaturing conditions (SDS-PAGE) versus native conditions (IF, IP) can yield different results.

• Verify antibody specificity in each system:

  • Perform validation controls (knockdown/knockout) in each experimental system.

  • Use multiple antibodies targeting different epitopes to confirm consistent patterns.

• Consider cell/tissue-specific expression patterns:

  • SMARCD1 expression varies across tissues and cell types, with notable expression in brain tissue .

  • Expression levels also change during cellular differentiation processes .

What common technical issues arise when working with SMARCD1 antibodies and how can they be resolved?

Common technical issues include:

• Weak or no signal:

  • Verify SMARCD1 expression in your experimental system before antibody detection.

  • Increase antibody concentration or incubation time.

  • For FFPE tissues, optimize antigen retrieval methods (TE buffer pH 9.0 is recommended for SMARCD1) .

  • Check antibody viability; improper storage can lead to degradation.

• Non-specific bands in Western blot:

  • Increase blocking stringency or duration.

  • Optimize antibody dilution (1:500-1:1000 is recommended) .

  • Increase washing steps in duration and number.

  • Use gradient gels to better resolve proteins in the 58 kDa range (observed molecular weight of SMARCD1) .

• Inconsistent immunoprecipitation results:

  • Optimize antibody amount (0.5-4.0 μg per 1.0-3.0 mg of total protein) .

  • Consider cross-linking antibody to beads to prevent antibody contamination in eluates.

  • Use gentle lysis and washing conditions to preserve protein complexes.

• High background in immunofluorescence:

  • Optimize antibody dilution (1:20-1:200 is recommended) .

  • Increase washing steps and duration.

  • Use appropriate blocking agents to reduce non-specific binding.

How should researchers approach experimental design to study dynamic changes in SMARCD1 expression during cellular differentiation?

Based on findings that SMARCD1 expression changes during cellular differentiation , researchers should:

• Design time-course experiments:

  • Collect samples at multiple time points during differentiation.

  • Use consistent protocols across time points to ensure comparability.

  • Include both early and late differentiation markers to correlate with SMARCD1 expression changes.

• Implement multiple detection methods:

  • Combine protein detection (Western blot, IF) with mRNA analysis (RT-qPCR).

  • Use flow cytometry for quantitative single-cell analysis of SMARCD1 alongside differentiation markers.

  • Apply ChIP-seq at different time points to track changes in SMARCD1 genomic localization.

• Include proper controls:

  • Compare normal differentiation to differentiation in SMARCD1 knockdown/overexpression systems.

  • Include undifferentiated cells as a baseline control at each time point.

  • Use appropriate housekeeping genes/proteins that remain stable during differentiation.

• Monitor SMARCD isoform switching:

  • Track expression of all three SMARCD isoforms simultaneously, as research shows SMARCD1 decreases while SMARCD3 increases during myeloid differentiation .

  • Use isoform-specific antibodies or primers to distinguish between family members.

• Correlate with functional outcomes:

  • Assess differentiation status using morphological analysis and surface marker expression.

  • Monitor changes in target gene expression known to be regulated by SMARCD1.

  • May-Grünwald Giemsa staining can be used to assess morphological changes associated with differentiation, as demonstrated in previous studies .