LSS Antibody

What is Lanosterol Synthase and why are antibodies against it important in research?

Lanosterol Synthase (LSS) is a key enzyme in the cholesterol biosynthesis pathway encoded by the LSS gene. In humans, the canonical LSS protein has 732 amino acid residues and a mass of 83.3 kDa . LSS belongs to the Terpene cyclase/mutase protein family and is also known as CTRCT44, HYPT14, OSC, 2,3-epoxysqualene-lanosterol cyclase, and APMR4 .

LSS antibodies are important research tools because they:

• Enable detection and quantification of LSS expression across different tissue types

• Support investigation of cholesterol biosynthesis dysregulation in disease states

• Allow for monitoring LSS localization and interactions with other proteins

• Provide insights into alternative splicing events that generate the 3 different reported isoforms

What are the common applications of LSS antibodies in research?

LSS antibodies have multiple research applications:

What is the subcellular localization of LSS and how does this impact antibody selection?

LSS is primarily localized in the endoplasmic reticulum (ER) . This subcellular localization has important implications for antibody selection and experimental design:

• For immunofluorescence studies, co-staining with ER markers (such as calnexin or PDI) is recommended to confirm proper localization

• Membrane permeabilization protocols must effectively target the ER membrane for optimal antibody access

• When studying potential non-canonical localizations of LSS, careful validation with multiple antibodies is essential to confirm unexpected findings

• For live cell imaging applications, consider the limitations of antibody penetration across the ER membrane

How can I validate the specificity of an LSS antibody for my experimental application?

Rigorous validation is essential before using an LSS antibody for critical experiments. The European Monoclonal Antibody Network recommends a stepwise validation approach :

• Positive and negative controls:

  • Use tissues/cells known to express LSS at high levels (widely expressed across tissue types )

  • Include knockout/knockdown samples as negative controls

  • Compare results with a second antibody targeting a different LSS epitope

• Epitope considerations:

  • Verify if the antibody targets Ro52 or Ro60 equivalent regions if these are relevant to your research

  • Antibodies raised against linear epitopes (peptide-based) work better for WB and IHC-P

  • Antibodies recognizing native epitopes perform better in IP, ELISA, and FCM

• Cross-reactivity testing:

  • Test antibody against recombinant LSS protein

  • Perform pre-adsorption tests with immunizing peptide (if available)

  • Verify species reactivity claims experimentally

• Technical validation:

  • Confirm the antibody produces a band of expected molecular weight (~83.3 kDa) in Western blots

  • For IHC/ICC, compare staining pattern with published literature

  • ELISA validation should include dilution linearity and spike recovery tests

What considerations are important when selecting between monoclonal and polyclonal LSS antibodies?

When working with alternatively spliced LSS isoforms, polyclonal antibodies that recognize multiple epitopes may detect all isoforms, while monoclonals might miss certain variants depending on the epitope location .

How should I optimize Western blotting protocols when using LSS antibodies?

Optimizing Western blot protocols for LSS detection requires consideration of several factors:

• Sample preparation:

  • As an ER-localized protein , use appropriate lysis buffers containing mild detergents (CHAPS, NP-40)

  • Include protease inhibitors to prevent degradation

  • For membrane proteins like LSS, avoid excessive heating during sample preparation

• Gel selection and transfer:

  • Use 8-10% acrylamide gels for optimal resolution of the 83.3 kDa LSS protein

  • Consider semi-dry transfer for 60-90 minutes or wet transfer overnight at 4°C

  • Use PVDF membranes for better protein retention and signal

• Blocking and antibody incubation:

  • 5% non-fat milk in TBST is typically effective for LSS antibodies

  • BSA-based blocking may be preferable for phospho-specific antibodies

  • Optimize primary antibody concentration (typically 1:500-1:2000)

  • Incubate at 4°C overnight for best results

• Detection and analysis:

  • Use appropriate HRP-conjugated secondary antibodies

  • For quantitative analysis, include loading controls (β-actin, GAPDH)

  • Validate signal linearity across a range of protein concentrations

• Troubleshooting:

  • For weak signals, increase antibody concentration or extend incubation

  • For high background, increase washing steps or reduce antibody concentration

What are the best practices for immunohistochemistry/immunocytochemistry when using LSS antibodies?

For optimal IHC/ICC results with LSS antibodies:

• Fixation and antigen retrieval:

  • Use 4% paraformaldehyde or formalin fixation

  • For paraffin sections, heat-mediated antigen retrieval in citrate buffer (pH 6.0) is typically effective

  • For frozen sections, acetone fixation for 10 minutes at -20°C often works well

• Blocking and permeabilization:

  • Block with 5-10% normal serum from the secondary antibody host species

  • For ER-localized LSS , effective permeabilization is crucial (0.1-0.3% Triton X-100)

  • Include 0.1% BSA in antibody diluent to reduce non-specific binding

• Antibody incubation:

  • Optimize antibody dilution (typically 1:100-1:500)

  • Incubate primary antibody overnight at 4°C or 1-2 hours at room temperature

  • Use fluorophore or enzyme-conjugated secondary antibodies based on detection method

• Controls and validation:

  • Include positive and negative tissue controls

  • Use isotype controls to assess non-specific binding

  • Consider dual staining with ER markers to confirm expected localization

How do I interpret conflicting results when using different LSS antibodies?

When facing conflicting results with different LSS antibodies:

• Epitope mapping:

  • Determine which protein regions each antibody targets

  • Consider if differences might reflect isoform-specific detection

  • Evaluate if some antibodies might recognize denatured versus native conformations

• Validation approach:

  • Use genetic models (knockdown/knockout) to verify specificity

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare results with orthogonal methods (qPCR, activity assays)

• Technical considerations:

  • Assess if conflicting results are technique-dependent

  • Review buffer compositions and experimental conditions

  • Consider the possibility of non-specific binding or cross-reactivity

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Employ complementary techniques to validate findings

  • Consult literature for reports of similar discrepancies

What approaches are available for quantitative analysis of LSS using antibody-based methods?

Several approaches can be used for quantitative analysis of LSS:

• Western blot densitometry:

  • Create standard curves using recombinant LSS protein

  • Ensure linear detection range by testing multiple exposures

  • Normalize to appropriate loading controls

• ELISA-based quantification:

  • Commercial ELISA kits for LSS are available

  • Develop sandwich ELISA using capture and detection antibodies

  • Consider the MASCALE method for absolute quantitation of antibody levels

• Flow cytometry:

  • Perform proper compensation when using multiple fluorophores

  • Use fluorescence minus one (FMO) controls

  • Calculate median fluorescence intensity (MFI) for population analysis

• Image-based quantification:

  • Use software like ImageJ for immunofluorescence quantification

  • Establish consistent acquisition parameters

  • Set appropriate thresholds and perform background correction

• Spatial analysis in tissues:

  • Quantify cell-type specific expression using multiplexed IHC

  • Measure subcellular distribution patterns

  • Compare expression levels across different tissue regions

How can deep learning approaches improve LSS antibody design and selection?

Deep learning is revolutionizing antibody research through several mechanisms:

• In-silico antibody generation:

  • Deep learning models can generate libraries of highly human antibody variable regions with desirable developability attributes

  • Models like Generative Adversarial Networks (GANs) and Wasserstein GAN with Gradient Penalty can produce novel antibody sequences

  • These approaches generate antibodies with high expression, monomer content, and thermal stability along with low hydrophobicity

• Epitope prediction and optimization:

  • Computational models predict optimal epitopes for targeting LSS

  • Machine learning algorithms enhance antibody affinity and specificity

  • In-silico screening reduces experimental validation requirements

• Performance prediction:

  • Models predict antibody performance in specific applications

  • Algorithms identify potential cross-reactivity issues

  • Computational approaches optimize antibody-antigen interactions

The ability to computationally generate developable human antibody libraries represents a significant advancement that may accelerate in-silico discovery of antibody-based biotherapeutics targeting proteins like LSS .

What role do LSS antibodies play in studying cholesterol biosynthesis disorders?

LSS antibodies are valuable tools for investigating cholesterol biosynthesis disorders:

• Disease mechanisms:

  • Detect alterations in LSS expression or localization in pathological states

  • Study LSS interactions with other cholesterol biosynthesis enzymes

  • Identify post-translational modifications affecting LSS function

• Diagnostic applications:

  • Develop immunoassays for measuring LSS levels in patient samples

  • Create tissue-based diagnostic tests using IHC

  • Develop multiplexed assays to analyze multiple pathway components

• Therapeutic monitoring:

  • Assess pharmacological modulation of LSS activity

  • Monitor changes in LSS expression during treatment

  • Evaluate the effects of novel cholesterol-lowering compounds

• Research applications:

  • Study LSS in cellular and animal models of cholesterol disorders

  • Investigate tissue-specific regulation of cholesterol biosynthesis

  • Explore non-canonical functions of LSS beyond cholesterol synthesis

How can I effectively use LSS antibodies in multiplex immunoassays?

Optimizing multiplex immunoassays with LSS antibodies requires:

• Antibody selection:

  • Choose antibodies raised in different host species for direct detection

  • Select isotype-diverse antibodies for isotype-specific secondary detection

  • Verify that antibodies don't compete for closely positioned epitopes

• Panel design:

  • Include relevant cholesterol pathway markers

  • Add subcellular markers to confirm ER localization

  • Consider including markers for cellular stress or lipid metabolism

• Technical optimization:

  • Test antibodies individually before combining

  • Confirm lack of cross-reactivity between detection systems

  • Optimize signal-to-noise ratio for each antibody

• Controls and validation:

  • Include single-stain controls

  • Use spectral unmixing for fluorescent multiplex assays

  • Validate results with orthogonal methods

• Analysis approaches:

  • Employ colocalization analysis for spatial relationships

  • Quantify relative expression levels across markers

  • Perform correlation analysis between pathway components

How can I address non-specific binding when using LSS antibodies?

Non-specific binding is a common issue that can be addressed through several approaches:

• Optimization strategies:

  • Increase blocking concentration (5-10% normal serum)

  • Add 0.1-0.5% non-ionic detergent to reduce hydrophobic interactions

  • Include 0.1-0.3% BSA in antibody diluent

  • Optimize antibody concentration using titration experiments

• Technical improvements:

  • Extend washing steps (number and duration)

  • Pre-adsorb antibody with tissue powder or cells lacking LSS

  • Filter antibody solution before use (0.22 μm)

  • Use more stringent washing buffers (higher salt concentration)

• Control experiments:

  • Include isotype controls to identify Fc-mediated binding

  • Use peptide competition assays

  • Perform staining on tissues known to lack LSS expression

• Alternative approaches:

  • Try a different LSS antibody targeting another epitope

  • Use more specific detection methods (e.g., tyramide signal amplification)

  • Consider directly conjugated primary antibodies to eliminate secondary antibody issues

What strategies can help optimize LSS antibody signal in challenging samples?

For challenging samples with low LSS expression or high background:

• Signal enhancement methods:

  • Implement heat-mediated antigen retrieval (citrate buffer pH 6.0)

  • Use signal amplification systems (avidin-biotin, tyramide)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try different detection systems (chromogenic vs. fluorescent)

• Sample preparation improvements:

  • Optimize fixation conditions (duration, temperature)

  • Test different permeabilization methods for ER access

  • Reduce autofluorescence using sodium borohydride or Sudan Black

  • Use antigen retrieval methods appropriate for ER proteins

• Advanced detection approaches:

  • Consider proximity ligation assay (PLA) for enhanced specificity

  • Use highly sensitive detection systems like QDs or organic dyes

  • Implement spectral imaging to separate signal from autofluorescence

• Technical considerations:

  • Optimize microscope settings (exposure, gain, offset)

  • Use deconvolution or confocal microscopy for improved resolution

  • Consider tissue clearing techniques for thick samples

How can I ensure reproducibility when working with LSS antibodies across different experimental batches?

Ensuring reproducibility requires systematic approach:

• Antibody management:

  • Use the same antibody clone and lot when possible

  • Maintain detailed records of antibody sources and lot numbers

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Store according to manufacturer recommendations

• Protocol standardization:

  • Develop detailed standard operating procedures (SOPs)

  • Maintain consistent reagent sources and preparation methods

  • Use automated systems where possible

  • Standardize timing of critical steps

• Quality control measures:

  • Include consistent positive and negative controls

  • Use internal reference standards

  • Implement quantitative acceptance criteria

  • Perform periodic validation experiments

• Data management:

  • Maintain comprehensive records of experimental conditions

  • Document any deviations from standard protocols

  • Implement blinding procedures for analysis

  • Use consistent image acquisition and analysis parameters

How are recent advances in antibody engineering improving LSS antibody performance?

Recent antibody engineering advances offer several benefits:

• Format modifications:

  • Species switching improves compatibility with secondary antibodies and enables easier co-labeling studies

  • Isotype and subtype switching alters in vivo effector function and stability

  • Engineering chimeric or humanized antibodies improves in vivo applications

• Affinity enhancements:

  • Directed evolution techniques increase binding affinity

  • Computational design optimizes complementarity-determining regions (CDRs)

  • Structure-guided engineering improves antigen recognition

• Stability improvements:

  • Engineering disulfide bonds enhances thermal stability

  • Reducing hydrophobic patches minimizes aggregation

  • Optimizing framework regions improves folding efficiency

• Functional modifications:

  • Adding site-specific conjugation sites for controlled labeling

  • Modifying Fc regions to alter effector functions

  • Developing bispecific formats for simultaneous targeting

The LS (Met428Leu and Asn434Ser) mutation in the Fc region has been shown to improve pharmacokinetic profiles by enhancing binding affinity to the neonatal Fc receptor, which could benefit LSS antibodies used in in vivo applications .

What emerging applications exist for LSS antibodies beyond traditional research techniques?

LSS antibodies are finding new applications:

• Advanced imaging techniques:

  • Super-resolution microscopy for nanoscale localization

  • Intravital imaging to study LSS dynamics in live animals

  • Correlative light and electron microscopy (CLEM) for ultrastructural analysis

• Single-cell analyses:

  • Mass cytometry (CyTOF) for high-dimensional single-cell profiling

  • Imaging mass cytometry for spatial proteomics

  • Single-cell western blotting for heterogeneity assessment

• Functional studies:

  • Antibody-mediated protein knockdown techniques

  • Intrabody approaches to study protein function

  • Proximity-dependent labeling to identify interacting partners

• Clinical applications:

  • Development of companion diagnostics

  • Therapies targeting LSS in cholesterol-related disorders

  • Biomarker discovery and validation

These emerging applications expand the utility of LSS antibodies beyond traditional protein detection into functional studies and potential therapeutic applications.