SCARA3 Antibody

What is SCARA3 and what are its key biological functions?

SCARA3, also known as scavenger receptor class A member 3, is a protein encoded by the human SCARA3 gene. The full-length protein has a molecular weight of approximately 65,137 daltons and exists in at least two identified isoforms. SCARA3 contains sites of glycosylation that may affect its function and detection . Functionally, SCARA3 plays critical roles in cellular homeostasis and oxidative stress response. It acts as a scavenger receptor involved in phagocytosis and immune response regulation by recognizing and clearing cellular debris and pathogens . Perhaps most significantly, SCARA3 has been identified as a protective factor against oxidative stress-induced cell death, particularly in cancer cells like those in multiple myeloma .

What detection methods are most effective for studying SCARA3 expression?

SCARA3 expression can be effectively detected using several complementary methods:

• Quantitative real-time PCR (qPCR): Effective for measuring SCARA3 mRNA expression levels using TaqMan gene qPCR expression assays, with 18S transcript used for normalization .

• Semi-quantitative RT-PCR: Useful for detecting different SCARA3 variants, particularly variant 2 (SCARA3 v2) .

• Western blot analysis: Provides protein-level detection using specific anti-SCARA3 antibodies, with a predicted band size of 66 kDa and 53 kDa, though the observed band is typically around 66 kDa .

• Immunohistochemistry (IHC): Allows for visualization of SCARA3 expression in tissue contexts, with recommended dilutions of 1:20-1:200 for optimal staining .

These methods can be used individually or in combination to provide comprehensive analysis of SCARA3 expression at both RNA and protein levels.

How can I differentiate between SCARA3 isoforms in my experiments?

Differentiating between SCARA3 isoforms requires strategic experimental approaches:

• Isoform-specific PCR primers: Design primers that span unique regions of each isoform. For SCARA3 variant 2, specific primers can be used in RT-PCR analysis as described in previous studies .

• Western blot analysis: The two SCARA3 isoforms have different molecular weights (predicted at 66 kDa and 53 kDa), which can be separated using appropriate polyacrylamide gel concentrations (10% reducing SDS-PAGE gel is recommended) .

• Antibody selection: Choose antibodies that can recognize epitopes specific to each isoform or use antibodies that detect common regions but can distinguish the isoforms by molecular weight differences in Western blots.

For definitive isoform identification, consider combining these approaches with mass spectrometry analysis to confirm the identity of detected proteins.

What are the optimal conditions for Western blot detection of SCARA3?

For optimal Western blot detection of SCARA3, the following protocol is recommended:

• Sample preparation: Extract total protein using standard lysis buffers containing protease inhibitors.

• Electrophoresis conditions: Use a 10% reducing SDS-PAGE gel to achieve appropriate separation of SCARA3 protein bands .

• Transfer parameters: Transfer proteins to PVDF membranes using standard transfer conditions.

• Blocking: Block non-specific binding with 5% skim milk in TBST buffer (4 mM Tris base, 10 mM NaCl, pH 7.5, 0.1% Tween-20) .

• Primary antibody: Incubate overnight at 4°C with anti-SCARA3 antibody at a dilution of 1:1000-1:5000 .

• Secondary antibody: Use an appropriate HRP-conjugated secondary antibody (e.g., goat polyclonal to rabbit IgG at 1/10000 dilution) .

• Detection: Develop using enhanced chemiluminescence assay for optimal visualization .

The observed band size for SCARA3 is typically around 66 kDa, which corresponds to one of its predicted isoforms .

How should I design experiments to study SCARA3 induction under oxidative stress?

To effectively study SCARA3 induction under oxidative stress conditions, consider the following experimental design:

• Oxidative stress inducers:

  • Ionizing radiation (IR): Use appropriate doses (research has shown SCARA3 induction at standard therapeutic doses)

  • Hydrogen peroxide (H₂O₂): Treatment with 200 μM for 6 hours has shown effective induction

  • Chemotherapeutic drugs: Dexamethasone (5 μM), Bortezomib (20 nM), or Arsenic trioxide (2 μM) for 12 hours

• Control conditions:

  • Include untreated cells as negative controls

  • Consider N-acetylcysteine (NAC, 10 mM) pre-treatment 1 hour before oxidative stress induction as an antioxidant control

• Time course analysis: Monitor SCARA3 expression at multiple time points (6h, 12h, 24h) to capture the dynamics of induction

• Detection methods:

  • qPCR for mRNA expression (see normalized fold-change calculation using 2⁻ᐩᐩCT)

  • Western blot for protein expression (optimal detection at 24h post-treatment)

This comprehensive approach will allow for robust characterization of SCARA3's response to various oxidative stressors.

What controls should be included when performing immunohistochemistry with SCARA3 antibodies?

When performing immunohistochemistry with SCARA3 antibodies, the following controls are essential:

• Positive tissue controls: Include tissues with known SCARA3 expression, such as liver or kidney tissue, which have been confirmed to express SCARA3 .

• Negative controls:

  • Primary antibody omission: Replace primary antibody with antibody diluent

  • Isotype control: Use matched concentration of non-specific rabbit IgG

  • Blocking peptide control: Pre-absorb the antibody with the immunizing peptide (recombinant Human SCARA3 protein, 201-466AA region)

• Dilution series: Test a range of antibody dilutions (1:20-1:200 is recommended) to determine optimal staining conditions .

• Cellular localization control: Compare staining pattern to known subcellular localization of SCARA3 to confirm specificity.

These controls will help validate the specificity of the observed staining and minimize the risk of false-positive or false-negative results.

How can SCARA3 expression be manipulated in experimental systems to study its function?

SCARA3 expression can be experimentally manipulated using several approaches:

• Overexpression systems:

  • Transfection with SCARA3 expression vectors in appropriate cell lines

  • Selection of stable cell lines overexpressing SCARA3 for long-term studies

  • This approach has been used to demonstrate that SCARA3 overexpression confers resistance to dexamethasone and bortezomib in myeloma cells

• Knockdown/knockout strategies:

  • SCARA3-specific lentiviral shRNAs have been successfully used to knockdown SCARA3 expression

  • CRISPR-Cas9 genome editing for complete knockout studies

  • Knockdown of SCARA3 has been shown to sensitize myeloma cells to chemotherapy-induced cell death

• Epigenetic modulation:

  • Treatment with 5-aza-2′-deoxycytidine (aza-dC, 2.5 μM for 48 h) to reverse DNA methylation

  • Trichostatin A (TSA, 100 nM for 24 h) to inhibit histone deacetylases

  • These treatments can reactivate epigenetically silenced SCARA3, as demonstrated in MM.1S myeloma cells

Each approach has specific advantages depending on your research question, with overexpression useful for gain-of-function studies and knockdown/knockout for loss-of-function analyses.

What is the relationship between SCARA3 expression and cancer progression, particularly in multiple myeloma?

The relationship between SCARA3 expression and cancer progression, particularly in multiple myeloma (MM), is complex and clinically significant:

This evidence suggests that SCARA3 functions as a protective factor against oxidative stress in MM cells and could serve as both a prognostic biomarker and a potential therapeutic target.

How might SCARA3's role in oxidative stress response be exploited for therapeutic purposes?

SCARA3's role in oxidative stress response presents several potential therapeutic strategies:

• Combination therapy approaches:

  • Inhibition of SCARA3 in combination with oxidative stress-inducing chemotherapies could enhance treatment efficacy

  • This approach has shown promise in experimental models, where SCARA3 knockdown sensitized myeloma cells to dexamethasone and bortezomib

• Targeted SCARA3 inhibition strategies:

  • Development of small molecule inhibitors targeting SCARA3 function

  • Antisense oligonucleotides or siRNA-based approaches for transient SCARA3 suppression

  • Antibody-drug conjugates targeting SCARA3-expressing cells

• Biomarker-guided therapy selection:

  • Patient stratification based on SCARA3 expression levels to predict response to oxidative stress-inducing therapies

  • Monitoring SCARA3 levels during treatment to predict therapy resistance

• Epigenetic modulation:

  • For cancers with epigenetically silenced SCARA3, epigenetic drugs might restore SCARA3 expression

  • This could potentially resensitize cancer cells to endogenous ROS-induced cell death pathways

These approaches reflect the complex dual nature of SCARA3 in cancer: while its expression correlates with better prognosis in multiple myeloma patients, its induction by oxidative stress can also promote therapy resistance.

What are common issues with Western blot detection of SCARA3 and how can they be resolved?

Common issues with Western blot detection of SCARA3 and their solutions include:

• Multiple or unexpected bands:

  • Issue: Detection of bands at unexpected molecular weights

  • Solution: Verify antibody specificity with positive and negative controls; consider antibody pre-absorption with immunizing peptide; optimize primary antibody concentration (recommended 1:1000-1:5000)

• Weak or no signal:

  • Issue: Insufficient protein detection

  • Solution: Increase protein loading; reduce antibody dilution; extend exposure time; verify protein transfer efficiency with Ponceau S staining; consider alternative lysis buffers for improved protein extraction

• High background:

  • Issue: Non-specific binding obscuring specific signals

  • Solution: Increase blocking time/concentration (5% skim milk in TBST is recommended) ; increase washing steps; decrease primary antibody concentration; use freshly prepared buffers

• Inconsistent detection between experiments:

  • Issue: Variable results across repeated experiments

  • Solution: Standardize protein extraction methods; use consistent positive controls; prepare master mixes for antibody dilutions; standardize exposure times

• Glycosylation interference:

  • Issue: Variable band sizes due to glycosylation of SCARA3

  • Solution: Consider enzymatic deglycosylation of samples before electrophoresis to obtain more consistent band patterns

Implementing these troubleshooting approaches should improve the reliability and consistency of SCARA3 detection in Western blot experiments.

How can I optimize immunohistochemical detection of SCARA3 in different tissue types?

Optimizing immunohistochemical detection of SCARA3 across different tissue types requires systematic adjustment of several parameters:

• Tissue fixation and processing:

  • Optimal fixation: 10% neutral buffered formalin for 24-48 hours

  • Consider testing different antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • For difficult tissues, test freshly frozen sections vs. FFPE samples

• Antibody optimization:

  • Perform antibody titration (recommended range: 1:20-1:200)

  • Test different incubation times and temperatures (overnight at 4°C vs. 1-2 hours at room temperature)

  • Consider signal amplification systems for low-expressing tissues

• Tissue-specific considerations:

  • High background tissues (e.g., liver): Increase blocking time and consider adding protein blockers (BSA, normal serum)

  • Tissues with endogenous peroxidase (e.g., kidney): Extend peroxidase blocking step

  • Tissues with high autofluorescence: Use chromogenic detection instead of fluorescence

• Validation approaches:

  • Compare staining patterns with known SCARA3 expression profiles

  • Use multiple SCARA3 antibodies targeting different epitopes

  • Correlate IHC results with other detection methods (Western blot, qPCR)

By systematically optimizing these parameters for each tissue type, researchers can achieve consistent and specific detection of SCARA3 across diverse experimental contexts.

What are the best practices for preserving SCARA3 antibody stability and functionality?

To maintain optimal SCARA3 antibody stability and functionality, researchers should follow these best practices:

• Storage conditions:

  • Store antibodies at recommended temperature (typically -20°C for long-term storage)

  • Maintain in appropriate buffer conditions (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative)

  • Avoid repeated freeze-thaw cycles by preparing small working aliquots

• Handling procedures:

  • Keep antibodies on ice when in use

  • Avoid contamination by using sterile pipette tips

  • Return to proper storage promptly after use

  • Centrifuge briefly before opening to collect solution at the bottom of the tube

• Working dilution preparation:

  • Prepare fresh working dilutions for each experiment

  • Use recommended diluents specific for each application (ELISA: 1:2000-1:10000, WB: 1:1000-1:5000, IHC: 1:20-1:200)

  • Allow antibodies to reach room temperature before opening to prevent condensation

• Quality control measures:

  • Include positive controls in each experiment to verify antibody performance

  • Monitor for changes in signal intensity or background over time

  • Document lot numbers and correlate with experimental outcomes

  • Consider implementing antibody validation tests annually for antibodies in long-term use

Adherence to these guidelines will help ensure consistent experimental results and extend the useful life of SCARA3 antibodies.

How can I design experiments to investigate SCARA3's role in the immune response?

To investigate SCARA3's role in immune response, consider these experimental approaches:

• Immune cell expression profiling:

  • Quantify SCARA3 expression across immune cell populations using flow cytometry and Western blot

  • Compare expression levels in resting vs. activated states of various immune cells

  • Analyze expression changes during different phases of immune response (acute vs. resolution)

• Functional assays:

  • Phagocytosis assays: Compare phagocytic capacity in cells with normal, overexpressed, or knocked-down SCARA3

  • ROS production: Measure oxidative burst in neutrophils or macrophages with modulated SCARA3 expression

  • Cytokine profiling: Assess how SCARA3 expression affects cytokine production patterns using multiplex assays

• In vivo immune challenge models:

  • Generate SCARA3 knockout or conditional knockout mice

  • Challenge with immune stimulants (LPS, poly I:C) or pathogens

  • Assess immune parameters including cell recruitment, cytokine production, and resolution of inflammation

• Co-immunoprecipitation studies:

  • Identify SCARA3 interaction partners in immune cells

  • Compare interactome under basal conditions versus inflammatory stimulation

  • Validate key interactions using proximity ligation assays or FRET

These approaches will provide comprehensive insights into SCARA3's immune regulatory functions across different contexts and cell types.

What approaches can be used to study the relationship between SCARA3 and other scavenger receptors?

To study the relationship between SCARA3 and other scavenger receptors, consider these methodological approaches:

• Comparative expression analysis:

  • Perform parallel qPCR analysis of multiple SR-A family members (SCARA1, SCARA2, SCARA3, SCARA4, SCARA5) using primers as described by DeWitte-Orr et al.

  • Create expression heat maps across different tissues and disease states

  • Use single-cell RNA sequencing to identify cells co-expressing multiple scavenger receptors

• Functional redundancy assessment:

  • Generate single and combinatorial knockdowns/knockouts of scavenger receptors

  • Compare phenotypic consequences in key functional assays

  • Determine if overexpression of one receptor can compensate for loss of another

• Ligand competition studies:

  • Identify shared and unique ligands between scavenger receptors

  • Perform competitive binding assays with labeled ligands

  • Determine binding kinetics and receptor preferences

• Structural comparative analysis:

  • Compare protein domains and structures between scavenger receptors

  • Identify conserved motifs that might indicate shared functions

  • Create chimeric receptors to map functional domains

These approaches will help delineate the unique and overlapping functions of SCARA3 relative to other members of the scavenger receptor family, providing insights into their collective role in cellular homeostasis and immune function.

How can multi-omics approaches be integrated to comprehensively study SCARA3 function?

Integrating multi-omics approaches for comprehensive SCARA3 functional analysis:

• Genomics integration:

  • Whole genome sequencing to identify SCARA3 genetic variants

  • GWAS analysis to correlate SCARA3 variants with disease phenotypes

  • ChIP-seq to map transcription factor binding sites in the SCARA3 promoter region

• Transcriptomics approaches:

  • RNA-seq before and after SCARA3 modulation to identify downstream gene networks

  • Single-cell RNA-seq to characterize cell-type specific SCARA3 expression patterns

  • Alternative splicing analysis to identify novel SCARA3 transcript variants

• Proteomics strategies:

  • Proximity-dependent biotin identification (BioID) to map SCARA3 protein-protein interactions

  • Phosphoproteomics to identify post-translational modifications of SCARA3

  • SILAC or TMT labeling to quantify proteome changes after SCARA3 modulation

• Metabolomics integration:

  • Targeted and untargeted metabolomics to identify metabolic pathways affected by SCARA3

  • Flux analysis using isotope-labeled metabolites to determine SCARA3's impact on metabolic rates

  • Lipidomics to investigate SCARA3's role in lipid metabolism and oxidative stress

• Computational integration:

  • Pathway enrichment analysis across multi-omics datasets

  • Network analysis to identify SCARA3-centered regulatory networks

  • Machine learning approaches to predict SCARA3 function from integrated datasets

This comprehensive multi-omics approach will provide unprecedented insights into SCARA3's functional roles across diverse biological contexts and disease states.