RS1 Antibody, Biotin conjugated

What is RS1 protein and what cellular functions does it serve?

RS1 (retinoschisin 1), also known as XLRS1 or X-linked juvenile retinoschisis protein, is a 224 amino acid secreted protein with a molecular weight of approximately 25.6 kDa. The protein is primarily localized to the cell membrane and is also secreted extracellularly . RS1 serves several critical functions in retinal physiology:

• Binds negatively charged membrane lipids, particularly phosphatidylserine and phosphoinositides

• Plays an essential role in cell-cell adhesion processes during retinal development

• Maintains normal structural integrity and function of the retina

• Absence of functional RS1 results in retinal cavities, synaptic dysfunction, reduced visual acuity, and increased susceptibility to retinal detachment

RS1 shows particularly high expression in the retina at the protein level, and research indicates it is required for proper retinal organization and function .

What are the key specifications of commercially available RS1 Antibody, Biotin conjugated?

The RS1 Antibody, Biotin conjugated typically has the following technical specifications:

This information is essential for determining experimental compatibility and appropriate application conditions .

What are the optimal storage and handling conditions for RS1 Antibody, Biotin conjugated?

To maintain antibody stability and functionality, researchers should adhere to the following storage and handling protocols:

• Store upon receipt at -20°C or -80°C for long-term stability

• Avoid repeated freeze-thaw cycles that can compromise antibody integrity

• The antibody is typically supplied in liquid form containing buffer components:

  • 50% Glycerol (cryoprotectant)

  • 0.01M PBS, pH 7.4 (stabilizing buffer)

  • 0.03% Proclin 300 (preservative)

• Ship at 4°C during transport, then aliquot and freeze upon arrival to minimize freeze-thaw cycles

• Consider creating small working aliquots to avoid repeatedly thawing the entire stock

Following these guidelines ensures maximum retention of antibody activity and specificity throughout your research timeline.

What basic experimental applications are supported by RS1 Antibody, Biotin conjugated?

The biotin-conjugated RS1 antibody supports multiple experimental applications with varying recommended dilution ranges:

• ELISA: Primary validated application for all commercially available versions

  • Methodology: Can be used as a capture or detection antibody in sandwich ELISA configurations

  • Advantage: Biotin conjugation allows amplification with streptavidin-enzyme systems

• Western Blot (WB): Some versions validated for use at 1:2000-1:10000 dilution

  • Expected band: ~26 kDa (observed molecular weight matches calculated)

  • Tested reactivity: Human, mouse, and rat retinal/eye tissue samples

• Immunohistochemistry (IHC): Validated products usable at 1:50-1:500 dilution

  • Most effective with antigen retrieval using TE buffer pH 9.0 or alternative citrate buffer pH 6.0

  • Successfully tested on mouse eye tissue sections

• Immunofluorescence (IF): Validated for paraffin-embedded sections at 1:50-1:500 dilution

  • Successfully demonstrated in mouse eye tissue samples

Each application requires appropriate optimization for specific experimental conditions and sample types.

How can I optimize immunodetection protocols when using RS1 Antibody, Biotin conjugated?

Optimizing protocols with RS1 Antibody requires systematic approach considering multiple variables:

For ELISA optimization:

• Conduct antibody titration experiments (1:1000 to 1:20000) to determine optimal working concentration

• Test different blocking agents (BSA, milk proteins, commercial blockers) to minimize background

• For biotinylated antibodies, implement a biotin blocking step if using tissues with endogenous biotin

• Evaluate the effect of incubation time (2-16 hours) and temperature (4°C, room temperature)

• Consider signal amplification using streptavidin-HRP/AP systems with different substrate combinations

For immunohistochemistry and immunofluorescence:

• Compare antigen retrieval methods - data indicates TE buffer pH 9.0 performs better than citrate buffer pH 6.0 for RS1 detection

• Optimize primary antibody concentration between 1:50-1:500 based on signal-to-noise ratio

• For biotin-conjugated antibodies, use avidin-biotin blocking kit to reduce endogenous biotin interference

• When working with retinal tissue specifically:

  • Test different fixation protocols (4% PFA, methanol, acetone)

  • Consider thickness of retinal sections (7-10 μm optimal for most applications)

  • Implement longer permeabilization steps for better antibody penetration

For Western blot:

• Test both reducing and non-reducing conditions

• Optimize transfer time and voltage for efficient protein migration at RS1's molecular weight (26 kDa)

• Evaluate membrane types (PVDF vs. nitrocellulose) for optimal signal

• Consider extended blocking (2-16 hours) to minimize background with retinal tissue extracts

Systematic optimization across these variables will maximize detection sensitivity and specificity.

What controls should be incorporated when using RS1 Antibody, Biotin conjugated for experimental validation?

Proper experimental design requires multiple controls to ensure reliable and interpretable results:

Positive controls:

• Known RS1-positive samples: human, mouse, or rat retinal tissue lysates or sections

• Recombinant human RS1 protein (similar to the immunogen region 24-224AA)

• Cell lines transfected with RS1 expression vectors

Negative controls:

• RS1 knockout tissues or cells (when available)

• Secondary antibody-only controls (omitting primary antibody)

• For biotin-conjugated antibodies specifically:

  • Streptavidin-only controls to evaluate endogenous biotin

  • Isotype control (rabbit IgG-biotin, non-targeting) at matching concentration

Specificity controls:

• Pre-adsorption/blocking with immunizing peptide

• For retinal tissues, comparison with known RS1 expression patterns

• Western blot verification of single band at expected molecular weight (26 kDa)

Reproducibility controls:

• Technical replicates across different experimental runs

• Biological replicates from multiple donors/samples

• Standard curves using recombinant protein for quantitative applications

Implementing this comprehensive control strategy ensures data integrity and facilitates proper interpretation of experimental outcomes.

How can RS1 Antibody, Biotin conjugated be effectively used in studying X-linked juvenile retinoschisis (XLRS)?

RS1 Antibody provides a powerful tool for investigating X-linked juvenile retinoschisis through multiple research approaches:

Disease mechanism investigation:

• Analyze RS1 protein expression and localization in patient-derived samples compared to controls

• Correlate RS1 protein levels with specific RS1 gene mutations reported in clinical studies

• Examine structural changes in retinal layers associated with RS1 deficiency using immunohistochemistry

Gene therapy assessment:

• Monitor RS1 protein expression following AAV8-RS1 gene augmentation therapy

• Quantify restoration of RS1 protein in treated versus untreated tissues

• Correlation analysis between RS1 protein levels and functional/structural improvements

• Analysis of antibody responses against AAV8 vector and RS1 protein itself - clinical trials showed no detectable antibody response against RS1 protein following gene therapy

Experimental methodologies:

• Tissue immunostaining to visualize schisis (splitting) in retinal layers in relation to RS1 expression

• Quantitative ELISA to measure RS1 protein concentration in vitreous or aqueous humor

• Western blot analysis to compare RS1 protein isoforms or degradation products

• Co-immunoprecipitation studies to identify binding partners involved in RS1 adhesion functions

Clinical-research correlation:

• Stratification of patients based on RS1 protein expression patterns

• Association studies between RS1 antibody-detected protein levels and clinical parameters

• Immunohistochemical analysis correlated with OCT imaging findings

The biotin conjugation offers additional advantages through signal amplification when studying tissues with potentially low RS1 expression levels.

What strategies can resolve troubleshooting challenges when using RS1 Antibody, Biotin conjugated?

Researchers may encounter various technical issues when working with RS1 Antibody. Here are systematic troubleshooting approaches:

High background issues:

• Implement more stringent blocking: Extend blocking time (2-16 hours) and test alternative blocking agents

• For biotin-conjugated antibodies: Use commercial avidin/biotin blocking kits to reduce endogenous biotin signals

• Increase wash duration and frequency between steps

• Dilute antibody further (start with manufacturer recommendations, then adjust as needed)

• For retinal tissue specifically: Implement autofluorescence quenching steps

Weak or absent signal:

• Verify antigen retrieval efficacy: Compare TE buffer pH 9.0 with citrate buffer pH 6.0

• Optimize antibody concentration: Test concentration range around manufacturer recommendation (1:50-1:500 for IHC/IF)

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

• For biotin-conjugated antibodies: Implement signal amplification with streptavidin-HRP/AP systems

• Verify sample integrity: Confirm RS1 presence with alternative methods

Non-specific banding on Western blots:

• Optimize blocking conditions (5% BSA vs. 5% milk)

• Increase antibody dilution (1:5000-1:10000)

• Test longer wash durations between incubation steps

• Consider membrane type (PVDF may give cleaner results than nitrocellulose for certain applications)

• Verify lysis buffer compatibility (avoid detergents that might disrupt epitope recognition)

Inconsistent results between experiments:

• Standardize sample processing: consistent fixation time and conditions

• Aliquot antibodies to avoid freeze-thaw degradation

• Implement internal controls for normalization

• Standardize image acquisition parameters

• Consider batch effects in experimental design

Systematic documentation of these variables will facilitate effective troubleshooting and protocol optimization.

How can RS1 Antibody be utilized to evaluate gene therapy outcomes in retinal disorders?

RS1 Antibody provides critical tools for assessing gene therapy efficacy in studies targeting RS1-related disorders:

Pre-clinical assessment:

• Quantitative analysis of RS1 protein restoration following gene transfer

• Spatial distribution mapping of newly expressed RS1 protein across retinal layers

• Correlation between RS1 protein expression levels and functional/structural recovery metrics

• Time-course studies to determine protein expression kinetics after treatment

Clinical trial monitoring:

• Evaluation of RS1 protein expression in accessible samples (if available)

• Assessment of anti-RS1 antibody development as potential adverse effect

  • Current clinical trial data indicates no detectable antibody response against RS1 protein in treated patients

  • This contrasts with dose-dependent increases in anti-AAV8 antibodies observed in the same patients

Methodological approaches:

• Quantitative ELISA using biotinylated RS1 antibody for protein quantification

• Immunohistochemistry to assess localization patterns (in pre-clinical models)

• Western blot for semi-quantitative analysis of RS1 expression levels

• Multi-parameter correlation between:

  • RS1 protein levels (antibody detection)

  • Functional outcomes (ERG, visual acuity)

  • Structural improvements (OCT imaging of schisis cavities)

Experimental considerations:

• Dose-response relationships: Current clinical trials tested three dosage groups (1e9, 1e10, and 1e11 vector genomes/eye)

• Effect of inflammation on protein expression: Higher doses showed correlation between antibody titers and inflammatory responses

• Transient versus sustained expression patterns

• Effect of pre-existing immunity on treatment outcomes: Pre-existing neutralizing antibodies to AAV8 may affect expression

This antibody enables critical molecular monitoring that complements clinical and electrophysiological assessments of treatment efficacy.

How can RS1 Antibody, Biotin conjugated be incorporated in multiple labeling experiments?

Implementing multiple labeling strategies with biotin-conjugated RS1 antibody requires careful experimental design:

Advantages of biotin conjugation for multiplexing:

• Flexible detection using streptavidin conjugated to various fluorophores or enzymes

• Strong biotin-streptavidin interaction enhances detection sensitivity

• Signal amplification capabilities for low-abundance targets

Sequential labeling strategies:

• Implement the following protocol sequence for double or triple labeling:

  • Apply RS1 antibody-biotin first

  • Detect with fluorophore-conjugated streptavidin (e.g., streptavidin-Alexa Fluor)

  • Block remaining biotin binding sites with unconjugated streptavidin

  • Apply second primary antibody (non-biotinylated)

  • Detect with species-specific secondary antibody with different fluorophore

Co-localization studies with relevant markers:

• RS1 with retinal cell markers:

  • Photoreceptor markers (rhodopsin, cone opsins)

  • Bipolar cell markers (PKC-α)

  • Müller cell markers (GFAP, glutamine synthetase)

• RS1 with extracellular matrix components to study adhesion properties

• RS1 with membrane lipid markers to investigate binding to phosphatidylserine and phosphoinositides

Technical considerations:

• Fluorophore selection: Choose spectrally distinct fluorophores to minimize bleed-through

• Order of application: Apply lower-abundance targets first for optimal detection

• Control experiments: Single-label controls are essential to validate multiple labeling specificity

• Signal separation: Consider spectral unmixing for closely overlapping signals

• Microscopy requirements: Confocal or super-resolution techniques may be necessary for co-localization analysis

These approaches enable comprehensive characterization of RS1 protein interactions and localization patterns in complex retinal tissues.

How does RS1 Antibody performance compare across different animal models of retinal disease?

RS1 Antibody shows variable performance characteristics across species commonly used in retinal disease modeling:

Species reactivity comparison:

• Human: Primary validated reactivity for most commercial antibodies

• Mouse: Validated reactivity in retina/eye tissue samples

• Rat: Validated reactivity in retina tissue samples

• Other species: Limited validation data available for bovine, frog, zebrafish, chimpanzee, and chicken models, though RS1 orthologs have been identified in these species

Detection sensitivity by application:

• Western blot performance:

  • Mouse eye tissue: Strong single band at 26 kDa

  • Rat retina tissue: Reliable detection at expected molecular weight

  • Human samples: Validated but limited comparative data

• Immunohistochemistry performance:

  • Mouse eye tissue: Excellent signal with TE buffer pH 9.0 antigen retrieval

  • Human samples: Validated but requires careful optimization

Model-specific considerations:

• RS1 knockout mouse models: Valuable negative controls for antibody validation

• Transgenic models expressing human RS1: May require antibodies that distinguish human from mouse RS1

• AAV-RS1 gene therapy models: Useful for measuring restoration of protein expression

Cross-reactivity analysis:

• Epitope conservation between species affects antibody performance

• Antibodies raised against human recombinant RS1 (24-224AA) show good cross-reactivity with mouse and rat proteins

• Western blot may show slight molecular weight variations between species

These comparative data facilitate appropriate model selection and experimental design based on antibody performance characteristics.

What are the methodological considerations for using RS1 Antibody in analyzing patient-derived samples?

Working with patient samples requires specific methodological adaptations compared to research models:

Sample acquisition and preparation:

• Retinal biopsies (rare): Immediate fixation critical for epitope preservation

• Vitreous fluid: Concentration methods may be needed for low-abundance detection

• Blood samples: Limited utility as RS1 is primarily expressed in retina

• iPSC-derived retinal organoids: Emerging patient-specific model system

Technical adaptations:

• Fixation protocols: Optimize to balance tissue morphology with epitope preservation

  • 4% PFA (12-24 hours maximum) recommended for most applications

  • Avoid over-fixation which can mask epitopes

• Antigen retrieval: TE buffer pH 9.0 shows superior performance to citrate buffer

• Background reduction: Implement stronger blocking protocols for clinical specimens

• Signal amplification: Leverage biotin-streptavidin interaction for enhanced sensitivity

Clinical correlation strategies:

• Sample stratification based on:

  • RS1 mutation type (missense vs. null mutations)

  • Disease severity (visual acuity, ERG findings)

  • Age of patients (progression markers)

• Quantitative analysis methods:

  • Digital image analysis for immunohistochemistry

  • Densitometry for Western blot quantification

  • Quantitative ELISA for protein measurement

Ethical and practical considerations:

• Limited availability of human retinal tissues

• Appropriate controls (age-matched, treatment-matched)

• Consistency in sample handling and processing

• Informed consent and ethical approvals

• Sample anonymization and data protection

These methodological adaptations maximize research value from limited and precious patient-derived materials.

How can RS1 Antibody be utilized in developing biomarkers for retinal disease progression?

RS1 Antibody enables multiple approaches for biomarker development in retinal diseases:

Quantitative biomarker applications:

• Measurement of RS1 protein levels in accessible fluids:

  • Vitreous humor (surgical samples)

  • Aqueous humor (minimally invasive sampling)

  • Tears (non-invasive but challenging detection limits)

• Correlation with disease progression metrics:

  • Optical coherence tomography (OCT) measurements of schisis cavities

  • Visual acuity changes

  • Electroretinogram (ERG) parameters

Biomarker validation strategies:

• Longitudinal studies correlating RS1 levels with disease progression

• Comparative analysis between:

  • Different RS1 mutations (genotype-phenotype correlations)

  • Age-matched controls and patients

  • Different stages of disease progression

• Statistical validation of biomarker sensitivity and specificity

Methodological approaches:

• Sandwich ELISA using biotin-conjugated antibody for detection

• Multiplex immunoassays combining RS1 with other retinal proteins

• Immunohistochemical scoring systems for research biopsies

• Digital quantification of immunofluorescence intensity

Translational applications:

• Treatment response monitoring in gene therapy trials

• Patient stratification for clinical trials

• Early detection of disease progression

• Prognostic indicator development

This biomarker development strategy leverages the specificity of RS1 antibody detection to create clinically relevant disease monitoring tools.

What role does RS1 Antibody play in evaluating immune responses to gene therapy interventions?

RS1 Antibody provides critical tools for monitoring potential immunological challenges in gene therapy:

Monitoring antibody development against therapeutic protein:

• In clinical trials, patients receiving AAV8-RS1 gene therapy showed:

  • No detectable antibody response against the RS1 protein at any time point following vector dosing

  • This contrasts with dose-dependent increases in neutralizing antibodies against the AAV8 vector capsid

• This favorable immunological profile supports continued development of RS1 gene therapy approaches

Methodological approaches for immune monitoring:

• ELISA-based detection of anti-RS1 antibodies in patient serum

• Comparison between pre-treatment and post-treatment samples

• Correlation between antibody development and:

  • Treatment efficacy

  • Inflammatory responses

  • Vector dosage (1e9, 1e10, and 1e11 vector genomes/eye were tested)

Immunological considerations in experimental design:

• Baseline assessment of pre-existing immunity

• Time-course monitoring following intervention

• Correlation between immune responses and clinical inflammation

• Strategies to mitigate immune responses:

  • Immunosuppressive regimens are being explored to enhance safety and efficacy

  • Localized administration (intravitreal) may reduce systemic exposure

Research and clinical implications:

• Favorable immunological profile of RS1 protein (low immunogenicity)

• Vector-related immunity as primary challenge

• Dose-dependent relationship between vector dose and antibody development

• Impact of pre-existing immunity on treatment outcomes

These immunological monitoring strategies are essential for developing safe and effective gene therapy approaches for RS1-related disorders.