SCARB1 Antibody

What is SCARB1 and why is it a significant research target?

SCARB1 (Scavenger Receptor Class B Member 1), also known as SR-BI, is an integral membrane protein encoded by the SCARB1 gene in humans. Its significance stems from its primary function as a receptor for high-density lipoproteins (HDL), facilitating the uptake of cholesteryl esters into cells, particularly in the liver and adrenal glands .

The protein plays a crucial role in reverse cholesterol transport, which drives the movement of cholesterol from peripheral tissues toward the liver for excretion. This process serves as a protective mechanism against atherosclerosis development, the principal cause of heart disease and stroke . Beyond lipid metabolism, SCARB1 has been implicated in pathogen recognition through interactions with mycobacteria and viral proteins, and it regulates vitamin E levels in tissues .

Recent research has revealed its role in cancer progression, particularly how SCARB1-containing extracellular vesicles can promote nasopharyngeal carcinoma metastasis by modulating macrophage function . This multifunctional nature makes SCARB1 a compelling target for diverse research applications.

What are the key considerations when selecting a SCARB1 antibody for research applications?

When selecting a SCARB1 antibody, researchers should consider:

• Target epitope: Determine whether N-terminal, C-terminal, or internal epitopes are more suitable for your application. Some antibodies, like the PB9502 antibody, target a synthetic peptide corresponding to a sequence at the C-terminus of mouse SCARB1 .

• Species reactivity: Verify cross-reactivity with your species of interest. SCARB1 antibodies vary in their reactivity profiles. For example, some antibodies react with human, mouse, and rat SCARB1, while others may have more limited species reactivity .

• Antibody format: Consider whether you need a polyclonal, monoclonal, or recombinant antibody based on your experimental needs:

  • Polyclonal antibodies offer broad epitope recognition but lower specificity

  • Monoclonal antibodies provide higher specificity but may be less robust to target protein modifications

  • Recombinant antibodies offer superior lot-to-lot consistency and continuous supply

• Validated applications: Ensure the antibody has been validated for your specific application. For instance, the SCARB1 (E9H4F) Rabbit mAb has been validated for Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence .

• Observed molecular weight: Note that the observed molecular weight of SCARB1 (~80-85 kDa) often differs from the calculated molecular weight (~57 kDa) due to post-translational modifications .

What are the standard applications for SCARB1 antibodies in research?

SCARB1 antibodies have been validated for multiple applications:

Each application requires specific optimization for the particular antibody and experimental conditions. For Western blotting, for example, researchers have successfully used anti-SCARB1 antibodies with reducing conditions on 5-20% SDS-PAGE gels, transferring to nitrocellulose membranes, and blocking with 5% non-fat milk in TBS .

How can I optimize immunohistochemistry protocols for SCARB1 detection in different tissue types?

Optimizing IHC for SCARB1 detection requires careful consideration of several parameters:

• Antigen retrieval: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has proven effective for SCARB1 detection in paraffin-embedded sections. This step is critical as it helps expose epitopes that may be masked during fixation .

• Blocking conditions: Use 10% goat serum to reduce non-specific binding. The blocking step should be performed after antigen retrieval and before primary antibody incubation .

• Primary antibody concentration and incubation: For optimal results, use approximately 2 μg/ml of rabbit anti-SCARB1 antibody with overnight incubation at 4°C. This extended incubation period at low temperature enhances specific binding while minimizing background .

• Secondary antibody selection: Peroxidase-conjugated anti-rabbit IgG with 30 minutes incubation at 37°C works effectively. The secondary antibody should match the host species of your primary antibody .

• Detection system: HRP-conjugated detection systems with DAB as the chromogen provide good visualization of SCARB1 expression. The intensity of staining can be used to grade expression levels from negative (1) to strongly positive (4) .

• Tissue-specific considerations:

  • Liver tissue: Shows strong SCARB1 expression due to its role in cholesterol metabolism

  • Adrenal gland: Also exhibits high expression levels

  • Nasopharyngeal tissue: May show variable expression based on pathological state

A semi-quantitative scoring system can be employed for analysis, where staining area is graded from 1-4 (0-25%, 26-50%, 51-75%, >75%) and intensity is graded from 1-4 (negative to strongly positive). The product of these scores (ranging from 1-16) can then categorize SCARB1 expression as low (1-8) or high (9-16) .

What are the critical factors for achieving reliable Western blot results when detecting SCARB1?

Successfully detecting SCARB1 via Western blotting requires attention to several critical factors:

• Sample preparation:

  • For tissue samples: Use 30 μg of tissue lysate per lane under reducing conditions

  • For cell samples: Ensure complete lysis with appropriate buffers containing protease inhibitors

  • For extracellular vesicles: Special isolation and purification protocols are needed

• Gel electrophoresis parameters:

  • Use 5-20% gradient SDS-PAGE gels for optimal separation

  • Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

  • These conditions allow proper separation of SCARB1, which has an observed molecular weight of ~80-85 kDa (despite calculated MW of ~57 kDa)

• Transfer conditions:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Efficient transfer is crucial for subsequent detection

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

  • Incubate with anti-SCARB1 antibody at 0.5 μg/mL overnight at 4°C

  • Wash thoroughly with TBS containing 0.1% Tween-20 (3 times, 5 minutes each)

  • Incubate with appropriate secondary antibody (e.g., goat anti-rabbit IgG-HRP) at 1:5000 dilution for 1.5 hours at room temperature

• Detection strategy:

  • Use enhanced chemiluminescent (ECL) detection systems

  • Expect to visualize SCARB1 at approximately 80-85 kDa

  • The discrepancy between observed (80-85 kDa) and calculated (57 kDa) molecular weights is due to post-translational modifications

• Validation controls:

  • Positive control: Liver tissue lysates which naturally express high levels of SCARB1

  • Negative control: Samples with confirmed low SCARB1 expression or knockdown samples

  • Loading control: Use standard housekeeping proteins (β-actin, GAPDH) to normalize expression levels

How can SCARB1 antibodies be employed to investigate its role in extracellular vesicle-mediated cancer progression?

Recent research has revealed SCARB1's role in extracellular vesicle (EV)-mediated cancer progression, particularly in nasopharyngeal carcinoma (NPC). Here are methodological approaches to investigate this phenomenon:

• Isolation and characterization of SCARB1-containing EVs:

  • Isolate EVs from cell culture supernatants or patient serum using ultracentrifugation or commercial isolation kits

  • Confirm EV isolation by transmission electron microscopy (TEM)

  • Validate SCARB1 presence in EVs by Western blotting using specific anti-SCARB1 antibodies

  • Quantify SCARB1 levels in EVs from cancer patients versus normal controls

• Co-culture experiments to evaluate EV-mediated effects:

  • Establish in vitro systems using macrophages (M0, M1, M2) co-cultured with SCARB1-containing EVs

  • Assess changes in macrophage phenotype and function after EV exposure

  • Monitor specific downstream targets (HAAO in M1 macrophages, CYP1B1 in M2 macrophages) using antibodies against these proteins

• Functional assays to assess macrophage responses:

  • For M1 macrophages: Measure reactive oxygen species (ROS) levels using fluorescent probes after exposure to SCARB1-EVs and pro-ferroptosis agents like RSL3

  • For M2 macrophages: Evaluate phagocytic capacity using latex beads or cancer cells after exposure to SCARB1-EVs

  • Use cell viability assays (CCK8) to assess macrophage survival

• In vivo metastasis models:

  • Establish animal models using cancer cells with SCARB1 knockdown

  • Administer purified EVs through tail vein injection

  • Compare metastatic burden between groups with and without macrophage depletion

  • Use immunofluorescence to confirm co-localization of SCARB1-mediated targets with specific macrophage markers (e.g., iNOS for M1, CD163 for M2)

• Validation through rescue experiments:

  • Knockdown SCARB1 in cancer cells and observe reduced EV-mediated effects

  • Restore these effects by introducing SCARB1-rich EVs

  • This approach confirms SCARB1's specific role in the observed phenomena

Research has shown that SCARB1-EVs promote cancer metastasis through dual mechanisms: increasing ferroptosis in anti-tumor M1 macrophages via HAAO upregulation while inhibiting phagocytosis in M2 macrophages through CYP1B1 regulation .

What approaches can be used to resolve discrepancies between observed and predicted molecular weights of SCARB1 in experimental results?

Researchers often encounter a significant discrepancy between the calculated molecular weight of SCARB1 (~57 kDa) and its observed size on Western blots (~80-85 kDa). Here are methodological approaches to address and understand this discrepancy:

• Glycosylation analysis:

  • Treat protein samples with deglycosylation enzymes (PNGase F or Endo H) before SDS-PAGE

  • Compare migration patterns before and after treatment

  • A shift to lower molecular weight after treatment confirms glycosylation contribution

• Denaturing conditions optimization:

  • Test different denaturing conditions (varying SDS concentrations, with/without reducing agents)

  • Compare migration patterns under different conditions

  • Persistent high molecular weight under stringent denaturing conditions suggests covalent modifications

• 2D gel electrophoresis:

  • Separate proteins first by isoelectric point, then by molecular weight

  • This can reveal different isoforms or post-translationally modified variants

  • Multiple spots at different positions but same molecular weight suggest charge-modifying modifications

• Mass spectrometry analysis:

  • Perform immunoprecipitation using anti-SCARB1 antibodies

  • Subject the purified protein to mass spectrometry

  • Compare observed mass with theoretical mass

  • Identify specific modifications and their locations

• Expression system comparison:

  • Express recombinant SCARB1 in different systems (bacterial, insect, mammalian)

  • Compare migration patterns

  • Bacterial expression will likely yield the unmodified form, while mammalian systems will reproduce native modifications

• Mutational analysis:

  • Generate mutants lacking potential modification sites

  • Compare migration patterns of wild-type versus mutant proteins

  • Shifts toward the theoretical weight in mutants can identify critical modification sites

The observed higher molecular weight is likely due to extensive post-translational modifications, including glycosylation, phosphorylation, and other covalent additions that increase the apparent molecular weight on SDS-PAGE gels .

How can I troubleshoot non-specific binding when using SCARB1 antibodies in immunohistochemistry?

Non-specific binding is a common challenge in immunohistochemistry with SCARB1 antibodies. Here's a systematic approach to troubleshoot this issue:

• Optimize blocking conditions:

  • Increase blocking time (from 1 hour to 2-3 hours)

  • Try different blocking agents (5-10% normal serum from the species of secondary antibody, BSA, commercial blocking solutions)

  • For particularly challenging tissues, consider using a combination of protein and detergent-based blocking

• Titrate primary antibody concentration:

  • Test a range of concentrations (0.5-5 μg/ml) to determine optimal signal-to-noise ratio

  • Based on published protocols, 2 μg/ml has proven effective for SCARB1 detection in adrenal gland tissue, but this may vary by tissue type

• Modify antibody incubation conditions:

  • Extend incubation time but reduce antibody concentration

  • Compare overnight incubation at 4°C versus shorter incubations at room temperature

  • Add 0.1-0.3% Triton X-100 to antibody diluent to improve penetration and reduce non-specific membrane binding

• Implement additional washes:

  • Increase number and duration of washes between steps

  • Use TBS with 0.1% Tween-20 instead of PBS for more effective removal of unbound antibody

• Consider epitope retrieval modifications:

  • Compare different retrieval methods (heat-induced versus enzymatic)

  • Adjust pH of retrieval buffer (try both acidic and basic conditions)

  • EDTA buffer at pH 8.0 has been successfully used for SCARB1 detection

• Include proper controls:

  • Negative control: Omit primary antibody while maintaining all other steps

  • Absorption control: Pre-incubate antibody with immunizing peptide

  • Positive control: Include tissue known to express SCARB1 (liver or adrenal gland)

  • Isotype control: Use non-specific IgG from the same species as primary antibody

• Consider signal amplification alternatives:

  • Compare direct HRP-conjugated systems versus avidin-biotin amplification

  • Explore tyramide signal amplification for low-abundance targets

  • Test different chromogens beyond DAB if background remains problematic

What strategies can resolve technical challenges when investigating SCARB1 in extracellular vesicles?

Investigating SCARB1 in extracellular vesicles (EVs) presents unique technical challenges. Here are methodological approaches to address these:

• EV isolation optimization:

  • Compare ultracentrifugation, density gradient, size exclusion chromatography, and commercial kits

  • Validate isolation by transmission electron microscopy (TEM) to confirm vesicle morphology

  • For SCARB1-specific studies, immunoaffinity capture using anti-SCARB1 antibodies can enrich for SCARB1-positive EVs

• Sample preparation for Western blotting:

  • Concentrate EV samples appropriately (10-30 μg protein per lane)

  • Use RIPA buffer with protease inhibitors for effective lysis

  • Consider non-reducing conditions as some epitopes may be reduction-sensitive

  • Load EV markers (CD63, CD9, CD81) as positive controls

• Quantification challenges:

  • Standardize EV quantification using nanoparticle tracking analysis or tunable resistive pulse sensing

  • Normalize SCARB1 expression to total EV protein or specific EV markers

  • Consider flow cytometry with EV capture beads for SCARB1 surface expression analysis

• Co-localization studies:

  • For immunofluorescence, immobilize EVs on poly-L-lysine coated slides

  • Use membrane-specific dyes (PKH26, DiI) to label EV membranes

  • Perform dual labeling with anti-SCARB1 antibodies and EV markers

  • Employ super-resolution microscopy for detailed localization

• Functional assay considerations:

  • When assessing EV effects on recipient cells (e.g., macrophages), standardize EV doses

  • Include controls for soluble factors by using EV-depleted supernatant

  • Consider tracking EVs with fluorescent membrane dyes to confirm uptake

  • Use SCARB1-depleted EVs (from knockdown cells) as negative controls

• Storage and stability:

  • Analyze fresh EVs when possible

  • For storage, maintain at -80°C with protease inhibitors

  • Avoid repeated freeze-thaw cycles which can affect SCARB1 epitope integrity

  • Validate antibody performance with stored versus fresh EV samples

• Distinguishing SCARB1 populations:

  • Use sucrose gradient fractionation to separate different EV populations

  • Perform immunoblotting for SCARB1 across fractions

  • This approach helps distinguish SCARB1 in different EV subtypes (exosomes vs. microvesicles)

How can I effectively design experiments to investigate SCARB1's dual role in lipid metabolism and pathogen recognition?

SCARB1's multifunctional nature requires carefully designed experiments to dissect its different roles. Here's a methodological framework:

• Cell type selection strategy:

  • For lipid metabolism: Hepatocytes, adrenocortical cells, or macrophage foam cells

  • For pathogen recognition: Macrophages, dendritic cells, or epithelial cell barriers

  • Consider using cell lines with SCARB1 knockout/knockdown for comparative studies

• Lipid metabolism experimental design:

  • Measure HDL binding and cholesteryl ester uptake using fluorescently labeled HDL

  • Assess cholesterol efflux using radiolabeled cholesterol

  • Quantify SCARB1 expression levels and correlate with functional readouts

  • Compare wild-type cells with SCARB1-depleted cells

• Pathogen interaction studies:

  • Challenge cells with pathogens reported to interact with SCARB1 (mycobacteria, certain viruses)

  • Use fluorescently labeled pathogens to assess binding to cells expressing different levels of SCARB1

  • Perform co-immunoprecipitation with anti-SCARB1 antibodies to identify pathogen ligands

  • Analyze downstream signaling pathways activated upon pathogen binding

• Domain-specific functional analysis:

  • Generate domain-specific mutants or chimeric proteins

  • Express mutants in SCARB1-null backgrounds

  • Assess which domains are critical for different functions

  • Use domain-specific antibodies to block particular functions without affecting others

• In vivo experimental approaches:

  • Create tissue-specific SCARB1 knockout models

  • Challenge with high-fat diet (for metabolism studies) or pathogens (for recognition studies)

  • Conduct parallel experiments in the same animals to assess potential interconnections

  • Use site-directed mutagenesis to create animals with selective functional deficits

• Multi-parameter flow cytometry:

  • Design panels with anti-SCARB1 antibodies alongside markers for:

    • Lipid metabolism (intracellular lipid staining, LDL receptors)

    • Pathogen recognition (pattern recognition receptors, activation markers)

  • This allows correlation of SCARB1 expression with different functional parameters at the single-cell level

• Interaction network analysis:

  • Perform immunoprecipitation with anti-SCARB1 antibodies followed by mass spectrometry

  • Identify protein interaction partners in different functional contexts

  • Build interaction networks to visualize how SCARB1 participates in different cellular processes

  • Validate key interactions using proximity ligation assays or FRET techniques

This comprehensive approach allows delineation of SCARB1's different functions while potentially revealing interconnections between its metabolic and immune roles.

How can SCARB1 antibodies be utilized in investigating the role of this receptor in cancer progression beyond nasopharyngeal carcinoma?

Recent findings on SCARB1's role in nasopharyngeal carcinoma metastasis open avenues for investigating its involvement in other cancer types. Here are methodological approaches:

• Cancer tissue expression profiling:

  • Use tissue microarrays spanning multiple cancer types

  • Apply validated anti-SCARB1 antibodies for immunohistochemistry

  • Develop a standardized scoring system (1-16 scale as described earlier)

  • Correlate expression with clinical parameters and outcomes

• Tumor microenvironment studies:

  • Perform multiplexed immunofluorescence using anti-SCARB1 antibodies alongside:

    • Tumor markers

    • Macrophage phenotype markers (M1: iNOS; M2: CD163)

    • Other immune cell markers

  • This reveals spatial relationships between SCARB1-expressing cells and immune components

• Mechanistic investigations in different cancer types:

  • Generate SCARB1 knockdown/overexpression models in various cancer cell lines

  • Assess alterations in:

    • Proliferation and invasion capacities

    • EV production and content

    • Cholesterol metabolism (which may affect membrane signaling platforms)

  • Compare effects across cancer types to identify tissue-specific versus universal mechanisms

• SCARB1-EV targeted interventions:

  • Develop antibodies that specifically block SCARB1 in EVs

  • Test their ability to prevent pro-metastatic changes in macrophages

  • Evaluate in preclinical models across multiple cancer types

  • Compare efficacy with other EV-targeting strategies

• Pharmacological modulation:

  • Screen compounds that modulate SCARB1 function or trafficking

  • Assess effects on EV composition and function

  • Test these compounds in preclinical cancer models

  • Use anti-SCARB1 antibodies to monitor target engagement

• Liquid biopsy applications:

  • Develop protocols to isolate and analyze SCARB1-positive EVs from patient blood

  • Create assays using anti-SCARB1 antibodies for EV capture

  • Correlate SCARB1-EV levels with disease progression across cancer types

  • Explore potential as biomarkers for treatment response

• Pathway integration analysis:

  • Investigate how SCARB1 interacts with established cancer pathways

  • Use phospho-specific antibodies to assess downstream signaling

  • Perform gene expression profiling after SCARB1 modulation

  • Identify cancer type-specific versus shared pathways

These approaches can systematically expand our understanding of SCARB1's role beyond NPC to other cancer types, potentially revealing new therapeutic targets and biomarkers.

What are the latest methodological advances for studying SCARB1 interaction with viral pathogens?

SCARB1's role in pathogen recognition, particularly with viral proteins, represents an emerging research area. Here are cutting-edge methodological approaches:

• High-resolution imaging techniques:

  • Utilize super-resolution microscopy (STORM, PALM) to visualize SCARB1-virus interactions at nanoscale resolution

  • Implement live-cell imaging with fluorescently tagged SCARB1 and viral components

  • Apply correlative light and electron microscopy to connect functional observations with ultrastructural details

• Protein-protein interaction advanced analysis:

  • Use biolayer interferometry with purified SCARB1 and viral proteins to determine binding kinetics

  • Implement hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Apply AlphaFold-based structural predictions to model SCARB1-viral protein complexes

  • Validate interactions using proximity ligation assays in infected cells

• CRISPR-based genetic screening:

  • Perform CRISPR activation/inhibition screens targeting SCARB1 and related genes

  • Assess viral entry and replication efficiency

  • Identify co-factors required for SCARB1-mediated viral recognition

  • Use anti-SCARB1 antibodies to validate findings at the protein level

• Domain-specific functional mapping:

  • Generate a panel of SCARB1 constructs with domain deletions or mutations

  • Express in cells lacking endogenous SCARB1

  • Challenge with different viruses to map domain-specific interactions

  • Use conformation-specific antibodies to assess structural changes upon viral binding

• Organoid and tissue models:

  • Establish organoid cultures from relevant tissues (liver, lung epithelium)

  • Manipulate SCARB1 expression using genetic approaches

  • Challenge with viruses and assess infection dynamics

  • Perform multiplex immunostaining to visualize SCARB1-virus co-localization in complex tissue architecture

• Single-cell analysis pipelines:

  • Implement single-cell RNA-seq in infected cultures

  • Correlate SCARB1 expression with viral load and cellular response signatures

  • Perform single-cell proteomics to assess SCARB1-dependent signaling

  • Use flow cytometry with anti-SCARB1 antibodies to isolate infection-relevant subpopulations

• In vivo infection models with SCARB1 modulation:

  • Develop tissue-specific or inducible SCARB1 knockout animal models

  • Challenge with relevant viral pathogens

  • Assess viral dissemination, replication, and host immune responses

  • Use anti-SCARB1 antibodies for immunohistochemical analysis of infected tissues

These methodological advances can provide deeper insights into how SCARB1 participates in viral pathogenesis, potentially leading to new therapeutic strategies targeting these interactions.

What are the most promising future directions for SCARB1 antibody development to advance research in both metabolism and cancer?

The multifunctional nature of SCARB1 suggests several promising directions for antibody development:

• Domain-specific antibodies:

  • Develop antibodies targeting specific functional domains of SCARB1

  • Create tools that can selectively inhibit lipid transport versus pathogen recognition

  • These would enable more precise dissection of SCARB1's multiple functions

• Conformation-specific antibodies:

  • Design antibodies that recognize specific conformational states of SCARB1

  • These could help identify active versus inactive receptor populations

  • Such tools would provide insights into receptor regulation mechanisms

• Post-translational modification-specific antibodies:

  • Develop antibodies that specifically recognize phosphorylated, glycosylated, or otherwise modified SCARB1

  • These would help correlate modifications with functional states

  • They could also explain the observed molecular weight discrepancies in Western blots

• Function-blocking antibodies:

  • Engineer antibodies that specifically block EV-associated SCARB1 functions

  • Create tools that prevent SCARB1-mediated effects on macrophages

  • These could serve as prototypes for therapeutic development

• Multiplexed imaging-compatible antibodies:

  • Develop antibody panels for simultaneous detection of SCARB1 with pathway components

  • Design conjugates compatible with multiplexed imaging technologies

  • These would enhance our understanding of SCARB1's contextual functions in complex tissues

• Bispecific antibody constructs:

  • Create bispecific antibodies targeting SCARB1 and relevant partners

  • These could help visualize or modulate specific interaction networks

  • They might also have therapeutic potential in redirecting immune responses

• Intrabodies and nanobodies:

  • Develop cell-permeable antibody derivatives for intracellular SCARB1 targeting

  • These could help track and modulate SCARB1 trafficking and function

  • They would provide new tools for live-cell imaging and functional perturbation

These advanced antibody tools would significantly enhance our ability to investigate SCARB1's diverse functions in metabolism, cancer progression, and pathogen interactions, potentially leading to novel diagnostic and therapeutic approaches.