SMARCAL1 Antibody, HRP conjugated

What is SMARCAL1 and why is it important in research?

SMARCAL1 is a 954 amino acid member of the SWI/SNF family of helicase and ATPase proteins, primarily localized in the nucleus. It plays crucial roles in ATP-dependent nucleosome remodeling, which is essential for regulating gene expression and maintaining chromatin structure. SMARCAL1 contains a conserved C-terminal SNF2 domain, a helicase ATP-binding domain, and two HARP domains, all vital for chromatin dynamics . Its significance in research stems from its implications in multiple biological processes, including DNA replication, repair, and transcription regulation. Furthermore, defects in SMARCAL1 are linked to Schimke immuno-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by renal dysfunction, spondyloepiphyseal dysplasia, and T cell immunodeficiency . Recent studies have also identified SMARCAL1 as a potential therapeutic target in cancer immunotherapy due to its association with immune infiltration across multiple cancer types .

What detection applications is the HRP-conjugated SMARCAL1 antibody suitable for?

The HRP-conjugated SMARCAL1 antibody is suitable for multiple laboratory applications requiring sensitive protein detection. This antibody format is particularly valuable for:

• Western Blotting (WB): Provides enhanced sensitivity without requiring secondary antibody incubation, allowing for direct detection of SMARCAL1 in protein lysates from mouse, rat, and human samples. The HRP conjugation enables direct visualization through chemiluminescent substrates .

• Immunohistochemistry (IHC): Enables direct visualization of SMARCAL1 in tissue sections without the need for secondary antibody incubation, reducing background and increasing specificity .

• Enzyme-Linked Immunosorbent Assay (ELISA): The HRP conjugation allows for quantitative measurement of SMARCAL1 in solutions or cellular extracts through colorimetric or chemiluminescent detection .

• Chromatin Immunoprecipitation (ChIP) Assays: Though requiring careful protocol optimization, HRP-conjugated antibodies can be used in ChIP experiments to investigate SMARCAL1's interactions with chromatin.

When selecting detection methods, researchers should consider the experimental objectives, sample type, and required sensitivity levels.

What validation steps should be performed before using SMARCAL1 antibody in research?

Before incorporating SMARCAL1 antibody into research protocols, thorough validation is essential to ensure reliable and reproducible results:

• Specificity Testing:

  • Western blot analysis comparing wildtype cells/tissues with SMARCAL1 knockout samples

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • Pre-absorption with recombinant SMARCAL1 protein to validate antibody specificity

• Cross-Reactivity Assessment:

  • Testing against samples from multiple species (mouse, rat, human) to confirm cross-reactivity as indicated by manufacturer

  • Evaluating potential cross-reactivity with other SWI/SNF family proteins through comparative blotting

• Functional Validation:

  • Immunofluorescence or immunohistochemistry to confirm expected nuclear localization pattern

  • Verification of detection in tissues known to express SMARCAL1 (e.g., kidney, lymphoid tissues)

• HRP Activity Confirmation:

  • Control experiments testing the HRP conjugate's enzymatic activity with appropriate substrates

  • Determination of optimal substrate incubation times and concentrations

• Limit of Detection Determination:

  • Serial dilution analysis to establish sensitivity thresholds

  • Comparison with unconjugated primary antibody plus secondary detection systems

Comprehensive validation ensures that experimental results accurately reflect SMARCAL1 biology rather than technical artifacts.

What are the optimal storage and handling conditions for HRP-conjugated SMARCAL1 antibody?

Maintaining antibody stability and functionality requires adherence to specific storage and handling protocols:

The HRP conjugate is particularly sensitive to oxidizing agents, heavy metals, and microbial contamination. Using sterile techniques during handling and avoiding exposure to these substances is crucial for maintaining enzymatic activity. Additionally, monitoring solution clarity before use can help identify potential denaturation or microbial contamination .

How can SMARCAL1 antibody be used to investigate its role in chromatin remodeling during replication stress?

SMARCAL1 plays a critical role in replication fork remodeling, and investigating this function requires sophisticated experimental approaches using SMARCAL1 antibody:

• Chromatin Immunoprecipitation (ChIP) at Replication Forks:

  • SMARCAL1 antibody can be used to perform ChIP followed by sequencing (ChIP-seq) to map genome-wide binding sites during replication stress.

  • For nascent DNA at replication forks, combining iPOND (isolation of Proteins On Nascent DNA) with SMARCAL1 immunoprecipitation can identify SMARCAL1's recruitment to specific DNA structures.

• Proximity Ligation Assays (PLA):

  • Using HRP-conjugated SMARCAL1 antibody alongside antibodies against RPA complexes can visualize their physical proximity at replication forks.

  • This approach can reveal how SMARCAL1 ubiquitylation affects its interaction with RPA-coated DNA, as studies show ubiquitylation impedes this interaction .

• SMARCAL1 Activity Visualization:

  • Immunofluorescence using SMARCAL1 antibody on cells treated with replication stress inducers (hydroxyurea, aphidicolin) can track its recruitment to stalled forks.

  • Research has shown that RFWD3 knockout leads to the appearance of bright punctate foci of SMARCAL1 colocalizing with RPA70 in UV-treated cells, indicating that ubiquitylation controls SMARCAL1 activity at replication forks .

• Chromatin Fractionation Analysis:

  • SMARCAL1 antibody can be used in western blotting of chromatin fractions to quantify its recruitment to chromatin during replication stress.

  • Studies show that UV-induced chromatin association of SMARCAL1 during S-phase is enhanced in RFWD3 knockout cells, suggesting that RFWD3-mediated ubiquitylation regulates SMARCAL1's chromatin association .

These approaches collectively provide insights into the "Goldilocks zone" of SMARCAL1 activity, where both too little and too much activity at replication forks can be detrimental to genome stability .

What methodological approaches can detect SMARCAL1 post-translational modifications?

Investigating SMARCAL1 post-translational modifications (PTMs) is crucial for understanding its regulation and function:

• Ubiquitylation Detection:

  • IP-Western Analysis: Immunoprecipitate SMARCAL1 using HRP-conjugated antibody followed by western blotting with ubiquitin antibodies

  • Mass Spectrometry: Identify specific ubiquitylation sites on SMARCAL1 after immunoprecipitation

  • Research has identified 15 ubiquitylation sites distributed across SMARCAL1's functional domains, with five sites within the HARP2-SWI/SNF ATPase functional core (K411, 431, 450, 570, 647)

• Phosphorylation Analysis:

  • Phospho-specific Antibodies: Use in conjunction with SMARCAL1 antibody to detect phosphorylated forms

  • Phosphatase Treatment: Compare SMARCAL1 migration patterns before and after phosphatase treatment

  • Studies indicate SMARCAL1 is regulated by ATM-, ATR-, and DNA-PK-dependent phosphorylation during replication stress

• PTM Dynamics During Replication Stress:

  • Time-Course Analysis: Use SMARCAL1 antibody to track modification patterns after inducing replication stress

  • Inhibitor Studies: Apply kinase or ubiquitin ligase inhibitors to investigate PTM mechanisms

  • Research shows that simultaneous inhibition of ATR and ATM decreases SMARCAL1 ubiquitylation, with strong suppression occurring only when ATM, ATR, and DNA-PK are co-inhibited

• PTM Impact on Protein Interactions:

  • Modified vs. Unmodified Pulldown Assays: Compare binding partners of modified and unmodified SMARCAL1

  • In Vitro Association Studies: Assess how modifications affect SMARCAL1's ability to bind RPA-ssDNA

  • Published data demonstrates that ubiquitylation of SMARCAL1 leads to strong decreases in its association with RPA-ssDNA

These techniques can help elucidate the complex regulatory network controlling SMARCAL1 activity, which appears to involve a cascade of phosphorylation and ubiquitylation events to fine-tune its function at stalled replication forks.

How can SMARCAL1 antibody be used to investigate correlations between SMARCAL1 expression and immune infiltration in cancer?

Recent research has revealed significant associations between SMARCAL1 expression and immune infiltration in various cancer types, suggesting its potential as a therapeutic target in cancer immunotherapy . Investigating these correlations requires specialized methodological approaches:

• Multiplex Immunohistochemistry (mIHC):

  • HRP-conjugated SMARCAL1 antibody can be used alongside immune cell markers (CD4, CD8, etc.) in sequential staining protocols.

  • This technique allows visualization of spatial relationships between SMARCAL1-expressing cells and infiltrating immune cells.

  • Studies have found significant relationships between SMARCAL1 expression and the degrees of CD4 T cell, CD8 T cell, and T helper cell infiltration in malignancies including Glioma, LUAD, LIHC, KIRC, and UCEC .

• Flow Cytometry Analysis:

  • Using fixed and permeabilized tumor samples, researchers can utilize SMARCAL1 antibody in combination with immune cell markers.

  • This approach enables quantitative assessment of SMARCAL1 expression levels in relation to specific immune cell populations.

  • Research has shown positive associations between SMARCAL1 and the infiltration levels of Type 2 T helper cells (R = 0.28) and Activated CD4 T cells (R = 0.26) in Glioma .

• Correlation Analysis with ESTIMATE Scoring:

  • SMARCAL1 antibody-based protein quantification can be correlated with ESTIMATE scores (Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data).

  • Higher scores of stromal and immune cells have been associated with elevated SMARCAL1 expression in Glioma and KIRC tissues .

• Tumor Mutation Burden (TMB) Correlation:

  • SMARCAL1 antibody-based expression data can be analyzed alongside TMB metrics.

  • Studies have demonstrated that SMARCAL1 expression and TMB level were positively correlated in Glioma (R = 0.16, P < 0.001), LUAD (R = 0.16, P < 0.001), and KIRC (R = 0.11, P < 0.05) .

These methodologies provide a comprehensive toolkit for investigating SMARCAL1's potential role in tumor immune microenvironment modulation and cancer immunotherapy responsiveness .

What experimental approaches can resolve contradictory findings in SMARCAL1 research?

Scientific research on SMARCAL1 has yielded some apparently contradictory findings, particularly regarding its impact on cancer prognosis and its regulatory mechanisms. Resolving these contradictions requires sophisticated experimental approaches:

• Cancer Type-Specific Analysis:

  • Comparative IHC Studies: Use HRP-conjugated SMARCAL1 antibody across diverse cancer tissue microarrays

  • Correlation with Clinical Outcomes: Match SMARCAL1 expression with survival data in specific cancer subtypes

  • Research has shown that elevated SMARCAL1 is linked to poor outcomes in Glioma, LUAD, and LIHC but correlates with better survival in KIRC, demonstrating context-dependent roles

• Genetic Background Influences:

  • Isogenic Cell Line Panels: Create isogenic cell lines with varying genetic backgrounds expressing similar SMARCAL1 levels

  • CRISPR-Cas9 Modifier Screens: Identify genetic factors that influence SMARCAL1 function

  • Studies suggest that SMARCAL1's role may be modified by tissue-specific factors and genetic background

• Post-Translational Modification Complexity:

  • Combined PTM Analysis: Investigate how multiple modifications (phosphorylation, ubiquitylation) interact

  • Time-Resolved Analysis: Track modification sequence during replication stress response

  • Research indicates a cascade of events where phosphorylation may serve as a primer for subsequent ubiquitylation

• Methodological Reconciliation:

  • In Vitro vs. In Vivo Discrepancies: Compare findings from in vitro ubiquitylation systems with cellular observations

  • Analysis of Technical Variables: Assess how antibody specificity, cell synchronization, and stress induction methods affect results

  • Studies show that while ubiquitylation doesn't affect SMARCAL1's fork regression activity in vitro, it significantly impacts its function in vivo by controlling RPA association

• Context-Dependent Activity:

  • Microenvironment Reconstitution: Recreate tissue-specific microenvironments to study contextual functions

  • Single-Cell Analysis: Use SMARCAL1 antibody in single-cell protein studies to identify cell state-dependent activities

  • Research emphasizes that SMARCAL1 activity must be maintained in a "Goldilocks zone" as both too little and too much activity at forks is deleterious for genome stability

These approaches can help reconcile contradictory findings by accounting for context-dependent functions and complex regulatory mechanisms governing SMARCAL1 activity.

What are the optimal protocols for using SMARCAL1 antibody in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) is crucial for investigating SMARCAL1's protein-protein interactions, particularly with replication and repair factors. The following protocol has been optimized for SMARCAL1 antibody Co-IP experiments:

• Cell Lysis and Extract Preparation:

  • Harvest 1-2 × 10^7 cells and wash twice with ice-cold PBS

  • Lyse cells in IP Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) supplemented with protease and phosphatase inhibitors

  • For nuclear proteins like SMARCAL1, include a nuclear extraction step using hypotonic buffer followed by nuclear lysis

  • Sonicate briefly (3 × 10 seconds) to disrupt nuclear membranes

  • Clarify lysate by centrifugation at 14,000 × g for 10 minutes at 4°C

• Pre-clearing and Antibody Binding:

  • Pre-clear lysate with 50 μL of Protein A/G agarose beads for 1 hour at 4°C

  • For HRP-conjugated SMARCAL1 antibody, dilute to 5 μg/mL in 1 mL of pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Note: When using HRP-conjugated antibodies, avoid using reducing agents in buffers as they can affect HRP activity

• Immunoprecipitation and Washing:

  • Add 50 μL of fresh Protein A/G agarose beads and incubate for 2 hours at 4°C

  • Wash beads 5 times with Wash Buffer (IP Lysis Buffer with reduced detergent concentration)

  • For detecting transient interactions, consider crosslinking cells with formaldehyde (1%, 10 minutes) before lysis

  • Research shows SMARCAL1 interacts with RPA complexes, and this interaction is affected by ubiquitylation

• Elution and Detection:

  • Elute proteins by boiling beads in 50 μL of 2× SDS sample buffer at 95°C for 5 minutes

  • For HRP-conjugated antibodies, use non-reducing conditions for the antibody heavy/light chain detection

  • Analyze by SDS-PAGE and western blotting

  • When probing for interaction partners, use specific antibodies against proteins of interest

• Controls and Validation:

  • Include isotype control antibody IP to identify non-specific interactions

  • Perform reverse Co-IP using antibodies against suspected interaction partners

  • Include input (5-10% of lysate), non-bound, and IP fractions in analysis

  • For RPA interactions, studies show ubiquitylation leads to strong decreases in SMARCAL1's association with RPA-ssDNA

This protocol has been optimized to preserve both stable and transient interactions while minimizing background, making it particularly suitable for studying SMARCAL1's dynamic interactions during replication stress responses.

How can researchers quantitatively assess SMARCAL1 ubiquitylation status?

Accurately quantifying SMARCAL1 ubiquitylation is essential for understanding its regulation during replication stress. The following methodological approaches provide quantitative assessment of this modification:

• In Vivo Ubiquitylation Assays:

  • Ubiquitin Pulldown: Express His-tagged ubiquitin in cells and purify ubiquitylated proteins under denaturing conditions

  • SMARCAL1 Immunoblotting: Probe purified material with SMARCAL1 antibody to detect ubiquitylated forms

  • Quantification: Use densitometry to calculate the ratio of ubiquitylated to total SMARCAL1

  • Research has identified at least 15 sites that can be modified by ubiquitin on SMARCAL1, distributed on solvent accessible lysine residues across various functional domains

• Mass Spectrometry-Based Quantification:

  • Sample Preparation: Immunoprecipitate SMARCAL1 using HRP-conjugated antibody, digest with trypsin

  • MS Analysis: Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Site Identification: Look for GG remnants on lysine residues indicating ubiquitylation

  • Quantification: Use label-free or isotope labeling techniques to quantify modification stoichiometry

  • Studies have utilized this approach to identify ubiquitylation sites within the HARP2-SWI/SNF ATPase functional core (K411, 431, 450, 570, 647)

• Fluorescence-Based Ubiquitylation Sensors:

  • FRET-Based Detection: Generate SMARCAL1-fluorescent protein fusions with ubiquitin-binding domains

  • Live-Cell Imaging: Monitor FRET signal changes during replication stress

  • Quantification: Calculate FRET efficiency as a measure of ubiquitylation

  • This approach can provide real-time, quantitative assessment of SMARCAL1 ubiquitylation dynamics

• In Vitro Ubiquitylation Reconstitution:

  • Reaction Setup: Combine purified SMARCAL1, E1, E2, RFWD3 (E3), and ubiquitin with ATP

  • Time-Course Analysis: Sample reaction at defined intervals to track modification progression

  • Quantification: Use western blotting with SMARCAL1 antibody to quantify modification rate

  • In vitro studies have shown that ubiquitylation of SMARCAL1 impedes its interaction with RPA-ssDNA

• Monitoring Ubiquitylation in Response to Kinase Inhibition:

  • Inhibitor Treatment: Apply ATM, ATR, and DNA-PK inhibitors individually or in combination

  • SMARCAL1 IP: Immunoprecipitate SMARCAL1 and analyze ubiquitylation

  • Quantification: Calculate fold-change in ubiquitylation relative to untreated controls

  • Research shows that simultaneous treatment with ATR and ATM inhibitors decreases SMARCAL1 ubiquitylation, with strong suppression when ATM, ATR, and DNA-PK are co-inhibited

These methodologies provide complementary approaches to quantitatively assess SMARCAL1 ubiquitylation, enabling researchers to elucidate its complex regulation during replication stress responses.

How might SMARCAL1 antibodies be utilized in developing cancer immunotherapy strategies?

Recent research has identified SMARCAL1 as a potential therapeutic target in cancer immunotherapy due to its associations with immune infiltration across multiple cancer types . Future research utilizing SMARCAL1 antibodies could advance this field in several ways:

• Biomarker Development for Immunotherapy Response:

  • Tissue Microarray Analysis: Using HRP-conjugated SMARCAL1 antibody to screen patient samples before immunotherapy

  • Correlation Studies: Linking SMARCAL1 expression patterns with response to immune checkpoint inhibitors

  • Multiplex Analysis: Combining SMARCAL1 detection with immune cell markers and checkpoint molecules

  • Research has already established correlations between SMARCAL1 expression and immune cell infiltration in multiple cancer types, suggesting its potential as a predictive biomarker

• Targeted Therapeutic Development:

  • Antibody-Drug Conjugates (ADCs): Modifying SMARCAL1 antibodies to deliver cytotoxic agents to cancer cells

  • Intracellular Antibody Delivery: Developing methods to deliver SMARCAL1-targeting antibodies intracellularly

  • Bispecific Antibodies: Creating constructs targeting both SMARCAL1-expressing cells and immune effectors

• Combination Therapy Optimization:

  • Sequential Therapy Protocols: Using SMARCAL1 antibody-based diagnostics to time immunotherapy with other treatments

  • Resistance Mechanism Studies: Investigating SMARCAL1's role in acquired resistance to immunotherapy

  • Research shows SMARCAL1 expression exhibits a negative correlation with HUMORAL_IMMUNE_RESPONSE, which may influence therapy effectiveness

• Cancer Type-Specific Approaches:

  • Differential Targeting Strategies: Developing cancer-specific protocols based on SMARCAL1's varied prognostic implications

  • Personalized Medicine: Using SMARCAL1 antibody diagnostics to guide treatment selection

  • Studies indicate that elevated SMARCAL1 is linked to poor outcomes in Glioma, LUAD, and LIHC but correlates with better survival in KIRC

• Mechanistic Understanding of Immune Modulation:

  • Signaling Pathway Analysis: Using SMARCAL1 antibodies to investigate downstream immune signaling

  • Tumor Microenvironment Studies: Assessing how SMARCAL1 affects the recruitment and function of immune cells

  • Genome Stability Connection: Exploring how SMARCAL1's role in maintaining genome stability influences anti-tumor immunity

These approaches represent promising directions for translating basic SMARCAL1 research into clinical applications, potentially expanding the effectiveness of cancer immunotherapy across multiple tumor types.

What emerging technologies might enhance the utility of SMARCAL1 antibodies in research?

As methodologies evolve, several emerging technologies hold promise for expanding the applications of SMARCAL1 antibodies in research:

• Spatial Transcriptomics and Proteomics Integration:

  • Combining HRP-conjugated SMARCAL1 antibody detection with spatial transcriptomics techniques

  • Creating multidimensional maps of SMARCAL1 protein expression alongside gene expression patterns

  • This integration could reveal spatial relationships between SMARCAL1 and immune cell infiltration in tumor microenvironments

• Single-Cell Protein Analysis:

  • Adapting SMARCAL1 antibodies for use in mass cytometry (CyTOF) and single-cell proteomics

  • Developing microfluidic platforms for single-cell western blotting with SMARCAL1 detection

  • These approaches could uncover cell-to-cell heterogeneity in SMARCAL1 expression and modification states

• Super-Resolution Microscopy Applications:

  • Utilizing fluorophore-conjugated SMARCAL1 antibodies in techniques like STORM and PALM

  • Enabling nanoscale visualization of SMARCAL1 localization at replication forks

  • This could provide unprecedented insights into how ubiquitylation affects SMARCAL1's association with RPA-coated DNA at the molecular level

• Functional Antibody Development:

  • Engineering antibodies that specifically recognize ubiquitylated vs. non-ubiquitylated SMARCAL1

  • Developing conformation-specific antibodies that detect active vs. inactive SMARCAL1 states

  • These tools could enable direct monitoring of SMARCAL1 regulation in living cells

• In Situ Proximity Labeling:

  • Coupling SMARCAL1 antibodies with enzymes like APEX2 or TurboID for proximity labeling

  • Mapping the dynamic interactome of SMARCAL1 during normal replication and stress conditions

  • This approach could identify novel interaction partners influencing SMARCAL1's "Goldilocks zone" of activity

• CRISPR-Based Genomic Tagging:

  • Using CRISPR/Cas9 to introduce epitope tags at the endogenous SMARCAL1 locus

  • Enabling antibody-based tracking of SMARCAL1 dynamics without overexpression artifacts

  • This strategy could provide more physiologically relevant insights into SMARCAL1 regulation

These technological advances promise to expand our understanding of SMARCAL1's complex functions in genome maintenance and its emerging roles in cancer and immunotherapy, potentially leading to novel diagnostic and therapeutic approaches.