smarcad1b Antibody

What is SMARCAD1B and how is it related to SMARCAD1?

SMARCAD1B is a DNA helicase paralog that demonstrates intrinsic ATP-dependent nucleosome-remodeling activity similar to SMARCAD1. While both proteins belong to the SWI/SNF family of chromatin remodelers, SMARCAD1B has distinct functional roles. SMARCAD1 plays critical roles in both DNA repair and chromatin remodeling, specifically through modulating histone H3K9me3 marks linked to gene repression. Both proteins share significant homology but operate in potentially different cellular contexts. Research suggests SMARCAD1 regulates chromatin accessibility at immune-related gene loci, including Il6 and Tnf. By contrast, SMARCAD1B's specific regulatory targets require further characterization, though it likely shares some functional overlap with SMARCAD1.

What are the validated applications for SMARCAD1B antibodies?

Most commercially available SMARCAD1B antibodies are validated primarily for Western blotting applications. Similar to the SMARCAD1 antibody (which is validated at 1:1000 dilution for Western blotting), SMARCAD1B antibodies typically detect proteins in the 150 kDa range. Some antibodies may have cross-validation for additional techniques including:

• Immunoprecipitation (IP)

• Immunofluorescence (IF)

• Chromatin immunoprecipitation (ChIP)

What are the optimal storage and handling conditions for SMARCAD1B antibodies?

SMARCAD1B antibodies, like most research antibodies, require specific storage conditions to maintain functionality. Based on similar antibody guidelines:

After reconstitution, antibodies should be stored in small aliquots at -20°C similar to the SMARCAD1 antibody recommendations. Repeated freeze-thaw cycles significantly reduce antibody activity and specificity, potentially leading to experimental inconsistencies.

What are the expected cross-reactivity profiles for SMARCAD1B antibodies?

Cross-reactivity assessments for SMARCAD1B antibodies should be carefully considered when designing experiments with multiple species. While antibodies may be raised against human SMARCAD1B, predicted reactivity with mouse or rat samples may remain unconfirmed experimentally. When selecting an antibody:

• Confirm species reactivity through literature or manufacturer validation

• Consider sequence homology between species (human SMARCAD1 antibodies typically have predicted 100% homology with primate samples)

• Perform validation experiments in your specific experimental system

• Be particularly cautious about cross-reactivity with SMARCAD1 and other SWI/SNF family members

How can I validate the specificity of a SMARCAD1B antibody?

Rigorous validation of SMARCAD1B antibody specificity requires multiple complementary approaches:

• Knockout/Knockdown Controls: Generate SMARCAD1B knockout or knockdown cells using CRISPR-Cas9 or siRNA approaches. Compare antibody signal between wild-type and knockout/knockdown samples to confirm specificity.

• Peptide Competition Assay: Pre-incubate the antibody with excess SMARCAD1B-specific peptide before application in Western blotting or immunostaining. Specific binding should be blocked by the peptide.

• Multiple Antibody Validation: Use at least two independent antibodies targeting different SMARCAD1B epitopes. Concordant results increase confidence in specificity.

• Mass Spectrometry Confirmation: Perform immunoprecipitation with the SMARCAD1B antibody followed by mass spectrometry to identify pulled-down proteins.

• Recombinant Protein Controls: Test antibody against purified recombinant SMARCAD1B and related proteins (especially SMARCAD1) to assess cross-reactivity.

This multi-faceted approach helps distinguish between specific SMARCAD1B detection and potential cross-reactivity with related chromatin remodelers, which is particularly important given the sequence homology between SMARCAD family members.

What is the recommended protocol for optimizing Western blotting with SMARCAD1B antibodies?

Optimizing Western blotting for SMARCAD1B (expected MW: ~150 kDa) requires careful attention to several parameters:

• Sample Preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if investigating phosphorylation states

  • Sonicate samples to shear DNA and release chromatin-bound proteins

• Gel Electrophoresis:

  • Use 6-8% SDS-PAGE gels to properly resolve high molecular weight proteins

  • Load 20-50 μg total protein per lane

  • Include molecular weight markers covering 100-250 kDa range

• Transfer Conditions:

  • Employ wet transfer systems for large proteins (>100 kDa)

  • Transfer at 30V overnight at 4°C for optimal results

  • Use PVDF membranes (0.45 μm pore size) for better protein retention

• Antibody Incubation:

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

  • Incubate with primary antibody at 1:1000 dilution at 4°C overnight

  • Wash thoroughly with TBST (4 × 5 minutes)

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour

• Detection Optimization:

  • Use enhanced chemiluminescent substrate appropriate for low-abundance proteins

  • Expose for multiple time intervals to determine optimal signal-to-noise ratio

  • Consider fluorescent secondary antibodies for improved quantification

Including both positive controls (cell lines with known SMARCAD1B expression) and negative controls is essential for accurate interpretation.

What are the best approaches for detecting SMARCAD1B in different cell types?

Detection strategies for SMARCAD1B must be tailored to specific cell types and experimental questions:

• Cell Line Selection:

  • SMARCAD1 expression has been characterized in intestinal epithelial cells

  • Consider cell lines relevant to chromatin remodeling research

  • Include controls with known SMARCAD1B expression levels

• Expression Assessment by Cell Type:

  • Begin with RT-qPCR to assess transcript levels across cell types

  • Follow with Western blotting to confirm protein expression

  • Use immunofluorescence to determine subcellular localization

• Tissue-Specific Considerations:

  • Fixation protocols may require optimization for different tissues

  • For intestinal epithelial cells, 4% paraformaldehyde fixation is typically effective

  • For brain tissue, consider alternative fixatives to preserve epitope accessibility

• Single-Cell Analysis:

  • Flow cytometry can be employed if developing a validated antibody for this application

  • Single-cell RNA-seq provides transcriptional data for SMARCAD1B expression

When investigating immune-related functions, consider that SMARCAD1 regulates genes involved in inflammatory responses, including Il6 and Tnf. Similar regulatory mechanisms may exist for SMARCAD1B, making immune cells particularly relevant for study.

How can I optimize chromatin immunoprecipitation (ChIP) protocols for SMARCAD1B?

Chromatin immunoprecipitation for SMARCAD1B requires special considerations due to its chromatin remodeling activity:

• Crosslinking Optimization:

  • Standard: 1% formaldehyde for 10 minutes at room temperature

  • For studying transient interactions: Add protein-protein crosslinkers (e.g., DSG)

  • Quench with 125 mM glycine for 5 minutes

• Chromatin Preparation:

  • Sonicate to achieve fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

  • Pre-clear chromatin with protein A/G beads and control IgG

• Immunoprecipitation Conditions:

  • Use 2-5 μg antibody per ChIP reaction

  • Incubate overnight at 4°C with rotation

  • Include input control and IgG negative control

  • Wash stringently to reduce background

• Analysis Approaches:

  • qPCR: Target known regulatory regions (promoters of immune genes like Il6 or Tnf)

  • ChIP-seq: Identify genome-wide binding sites and motifs

  • CUT&RUN or CUT&Tag for improved signal-to-noise ratio

• Data Interpretation:

  • Expected enrichment at regions with H3K9me3 marks (based on SMARCAD1 data)

  • Look for co-occupancy with other chromatin remodelers

  • Connect binding sites to gene expression changes

When analyzing results, remember that SMARCAD1 represses innate immune genes under steady-state conditions, with knockout models showing 2.5–4.5-fold upregulation of genes like Il6 and Tnf. SMARCAD1B may have similar or complementary regulatory functions.

How can I differentiate between SMARCAD1 and SMARCAD1B in my experiments?

Distinguishing between these related proteins requires careful experimental design:

• Antibody Selection:

  • Use antibodies raised against unique epitopes not shared between SMARCAD1 and SMARCAD1B

  • Validate specificity through knockout controls for each protein

  • Consider using epitope-tagged constructs when antibodies lack specificity

• Transcript Analysis:

  • Design qPCR primers targeting divergent regions

  • Use transcript-specific siRNAs for selective knockdown

  • Perform RNA-seq to quantify expression levels of both genes

• Functional Separation:

  • SMARCAD1 mediates responses to specific gut microbiota, especially TM7 phylum bacteria

  • Compare phenotypes after selective depletion of each protein

  • Assess differential response to stimuli (e.g., DNA damage, inflammatory signals)

• Protein Complex Analysis:

  • Immunoprecipitate each protein separately and analyze binding partners

  • Compare chromatin association patterns through ChIP-seq

  • Assess ATP-dependent remodeling activities in vitro

Creating a table of distinguishing features can help design experiments that effectively differentiate these related proteins.

What knockout or knockdown strategies are most effective for studying SMARCAD1B function?

Multiple approaches can be employed to deplete SMARCAD1B for functional studies:

• CRISPR-Cas9 Gene Editing:

  • Design multiple sgRNAs targeting early exons

  • Screen clones by Western blotting and genomic sequencing

  • Create conditional knockout systems (e.g., floxed alleles with Cre recombinase)

  • Consider creating epitope-tagged knockin lines for antibody-independent detection

• RNA Interference:

  • siRNA pools targeting multiple regions enhance knockdown efficiency

  • Validate knockdown by qRT-PCR and Western blotting

  • Optimize transfection conditions for each cell type

  • Use inducible shRNA systems for temporal control

• Antisense Oligonucleotides:

  • Design gapmers targeting SMARCAD1B-specific sequences

  • Optimize delivery methods for your experimental system

  • Monitor gene expression over time to determine knockdown kinetics

• Experimental Controls:

  • Include non-targeting controls

  • Rescue experiments with exogenous expression to confirm specificity

  • Monitor potential compensation by SMARCAD1 or other family members

When designing knockout experiments, consider that complete deletion of chromatin remodelers may cause developmental defects or lethality, as observed with related proteins like SMARCB1 and NURF complex components .

What experimental approaches can reveal SMARCAD1B interactions with chromatin?

Understanding SMARCAD1B's chromatin interactions requires specialized techniques:

• In Vitro Biochemical Assays:

  • Nucleosome sliding assays using reconstituted chromatin

  • ATPase activity measurements with nucleosome substrates

  • DNA binding and unwinding assays to characterize helicase activity

  • In vitro reconstitution of SMARCAD1B-containing complexes

• Chromatin Association Mapping:

  • ChIP-seq to identify genome-wide binding sites

  • CUT&RUN or CUT&Tag for improved resolution

  • ATAC-seq to assess chromatin accessibility changes following SMARCAD1B depletion

  • HiChIP to identify long-range chromatin interactions

• Histone Modification Analysis:

  • Based on SMARCAD1's role in modulating H3K9me3 marks, assess similar histone modifications

  • Perform sequential ChIP (re-ChIP) to identify co-occurrence with specific marks

  • Use mass spectrometry to identify histone modifications affected by SMARCAD1B

• Live-Cell Imaging:

  • FRAP (Fluorescence Recovery After Photobleaching) to assess chromatin binding dynamics

  • Single-particle tracking to monitor SMARCAD1B movement on chromatin

  • Optogenetic approaches to induce chromatin recruitment

These approaches can reveal how SMARCAD1B's ATP-dependent nucleosome-remodeling activity contributes to gene regulation and DNA repair processes.

How should I design experiments to investigate SMARCAD1B's role in inflammatory responses?

Based on SMARCAD1's established role in regulating inflammatory responses, similar studies for SMARCAD1B should consider:

• Cell System Selection:

  • Intestinal epithelial cells show significant SMARCAD1-dependent regulation

  • Consider both immune cells and epithelial barriers

  • Compare wild-type and SMARCAD1B-deficient systems

• Inflammatory Stimulation Protocols:

  • LPS treatment (10-1000 ng/ml, 2-24 hours)

  • TNF-α stimulation (10-50 ng/ml)

  • IL-1β exposure (10-20 ng/ml)

  • Microbial challenge (particularly TM7 phylum bacteria)

• Readout Selection:

  • qRT-PCR for inflammatory cytokines (Il6, Tnf, antimicrobial peptides)

  • ELISA for secreted cytokines

  • RNA-seq for genome-wide transcriptional changes

  • ChIP-seq before and after stimulation

• In Vivo Models:

  • DSS-induced colitis model (as used for SMARCAD1 studies)

  • Tissue-specific conditional knockouts

  • Bone marrow chimeras to distinguish hematopoietic vs. stromal contributions

• Mechanistic Investigations:

  • Assess chromatin accessibility changes at inflammatory gene loci

  • Measure H3K9me3 marks at candidate regulatory regions

  • Identify transcription factor binding affected by SMARCAD1B status

SMARCAD1 knockout models show 2.5–4.5-fold upregulation of inflammatory genes like Il6 and Tnf, providing quantitative benchmarks for comparison with SMARCAD1B studies.

How can I resolve common issues with SMARCAD1B antibody Western blotting?

Western blotting for large proteins like SMARCAD1B (~150 kDa) often presents technical challenges:

For high molecular weight proteins like SMARCAD1B, consider using gradient gels (4-15%) and extending transfer times to ensure complete transfer to the membrane.

What controls are essential for validating SMARCAD1B antibody specificity in immunofluorescence?

Rigorous immunofluorescence controls ensure reliable SMARCAD1B detection:

• Primary Controls:

  • SMARCAD1B knockout/knockdown cells (negative control)

  • SMARCAD1B overexpression system (positive control)

  • Peptide competition control (pre-incubation with immunizing peptide)

• Secondary Controls:

  • Secondary antibody only (background assessment)

  • Isotype control (non-specific binding evaluation)

  • Auto-fluorescence control (unstained sample)

• Fixation Optimization:

  • Compare multiple fixation methods (PFA, methanol, acetone)

  • Test antigen retrieval methods if nuclear antigens are masked

  • Optimize permeabilization conditions for nuclear proteins

• Co-localization Controls:

  • Use markers for nuclear compartments (DAPI for DNA)

  • Test co-localization with known interacting partners

  • Include markers for expected subcellular localization

• Image Acquisition Controls:

  • Standardize exposure settings across samples

  • Use identical processing parameters for experimental and control images

  • Implement blinded analysis to prevent bias

These controls help distinguish specific SMARCAD1B staining from technical artifacts or cross-reactivity with related chromatin remodelers.

How can I optimize antigen retrieval for SMARCAD1B immunohistochemistry in tissue sections?

Nuclear proteins involved in chromatin remodeling often require specialized antigen retrieval:

• Heat-Induced Epitope Retrieval (HIER):

  • Citrate buffer (pH 6.0): 95-100°C for 20 minutes

  • EDTA buffer (pH 9.0): Often superior for nuclear antigens like chromatin remodelers

  • Tris-EDTA (pH 9.0): Test against citrate for optimal results

• Pressure Cooking Methods:

  • High-pressure treatment (110-120°C) for 5-10 minutes

  • Allow 20-minute cool-down period before continuing protocol

  • Compare to microwave and water bath methods for your specific tissue

• Enzymatic Retrieval:

  • Proteinase K (10-20 μg/ml, 10-15 minutes at 37°C)

  • Trypsin (0.05-0.1%, 10-20 minutes at 37°C)

  • May be used alone or in combination with HIER

• Tissue-Specific Considerations:

  • Brain tissue: Additional permeabilization may be needed

  • Intestinal tissue: Reduce background with additional blocking

  • Formalin-fixed tissues: Extend retrieval times

• Protocol Optimization:

  • Test multiple retrieval methods side-by-side

  • Vary incubation times and temperatures

  • Consider dual retrieval approaches for challenging samples

Thorough optimization of antigen retrieval is crucial for studying nuclear proteins like SMARCAD1B, particularly in fixed tissue specimens.

How can single-cell approaches be applied to study SMARCAD1B function?

Single-cell methodologies offer unique insights into SMARCAD1B biology:

• Single-Cell RNA-Sequencing:

  • Assess cellular heterogeneity in SMARCAD1B expression

  • Identify cell populations with distinct SMARCAD1B-dependent gene programs

  • Compare transcriptional profiles before and after stimulation

• Single-Cell ATAC-Seq:

  • Map chromatin accessibility changes in SMARCAD1B-deficient cells

  • Identify cell type-specific regulatory elements

  • Link accessibility changes to transcriptional outcomes

• Single-Cell Western Blotting:

  • Quantify SMARCAD1B protein levels at single-cell resolution

  • Correlate with functional outcomes

  • Assess technical variability vs. biological heterogeneity

• CyTOF/Mass Cytometry:

  • Develop metal-conjugated antibodies against SMARCAD1B

  • Simultaneously measure multiple proteins in the same pathway

  • Characterize signaling networks dependent on SMARCAD1B

• Live-Cell Single-Molecule Tracking:

  • Monitor SMARCAD1B-chromatin interactions in real time

  • Measure residence times at specific genomic loci

  • Assess protein dynamics during cellular responses

These approaches can reveal heterogeneity in SMARCAD1B function and identify regulatory relationships that might be masked in bulk population studies.

What are the potential applications of SMARCAD1B antibodies in studying disease mechanisms?

SMARCAD1B antibodies can facilitate research into several disease contexts:

• Cancer Research:

  • Assess SMARCAD1B expression in tumor vs. normal tissue

  • Investigate correlation with chromatin dysregulation

  • Study potential roles in DNA damage repair pathways

• Inflammatory Conditions:

  • Based on SMARCAD1's role in colitis, investigate SMARCAD1B in inflammatory bowel disease

  • Study regulation of inflammatory gene expression

  • Assess potential as a biomarker for disease activity

• Neurodevelopmental Disorders:

  • Examine SMARCAD1B expression in neurodevelopmental tissues

  • Investigate potential overlap with SMARCA1 in neuronal development

  • Study interaction with other chromatin remodelers implicated in neurodevelopment

• Immunological Dysfunction:

  • Investigate antimicrobial peptide regulation (e.g., Reg3g, Pla2g2a)

  • Study impact on innate immunity gene expression

  • Assess contribution to host-microbiome interactions

• Therapeutic Target Validation:

  • Use antibodies to validate knockdown efficiency in therapeutic development

  • Monitor target engagement in drug screening

  • Develop proximity-based assays for compound screening

These applications leverage antibodies as tools for understanding SMARCAD1B's contribution to pathological mechanisms and potential therapeutic opportunities.

What are the current knowledge gaps in SMARCAD1B research?

Despite advances in chromatin remodeling research, several key questions about SMARCAD1B remain unanswered:

• Functional Distinction: How does SMARCAD1B's function differ from SMARCAD1, particularly in immune regulation?

• Protein Complexes: What are the unique binding partners of SMARCAD1B compared to other SMARCA family members?

• Tissue-Specific Roles: Does SMARCAD1B show tissue-specific expression or function different from SMARCAD1?

• Disease Relevance: Is SMARCAD1B dysregulation associated with specific pathologies, similar to SMARCAD1's role in colitis resistance?

• Regulatory Mechanisms: What controls SMARCAD1B expression and activity in different cellular contexts?

• Therapeutic Potential: Could targeting SMARCAD1B offer therapeutic benefits in inflammatory or neoplastic conditions?

Addressing these knowledge gaps represents an important frontier in chromatin biology research and will require continued development and characterization of specific SMARCAD1B research tools, including highly specific antibodies.

What emerging technologies might enhance SMARCAD1B antibody applications?

Several cutting-edge technologies promise to expand SMARCAD1B research capabilities:

• Proximity Labeling:

  • BioID or TurboID fusion proteins to identify interacting partners

  • APEX2-based approaches for temporal control of labeling

  • CUT&Tag for improved chromatin interaction mapping

• Nanobody Development:

  • Single-domain antibodies for improved access to epitopes

  • Cell-permeable nanobodies for live-cell applications

  • Multimodal detection with fluorescent nanobody fusions

• Spatial Transcriptomics:

  • Combined protein and RNA detection in tissue sections

  • Assessment of SMARCAD1B localization and function in complex tissues

  • Correlation with gene expression patterns at single-cell resolution

• Antibody Engineering:

  • Bispecific antibodies targeting SMARCAD1B and interacting proteins

  • Split-protein complementation for interaction studies

  • Antibody-based degradation systems (PROTAC approach)

• CRISPR-Based Technologies:

  • CUT&RUN/CUT&Tag methods using CRISPR-based targeting

  • dCas9-fusion proteins for site-specific chromatin modulation

  • Base editing for specific mutation introduction