CELSR1 Antibody, Biotin conjugated

What is CELSR1 and why is it significant in research?

CELSR1 (Cadherin EGF LAG seven-pass G-type receptor 1) is a receptor protein that plays a crucial role in cell-to-cell signaling during nervous system formation. This transmembrane protein belongs to the cadherin superfamily and is known by several aliases including Cadherin family member 9 (CDHF9), Flamingo homolog 2 (hFmi2), and FMI2 . With UniProt accession number Q9NYQ6, CELSR1 is primarily studied in neuroscience research contexts focusing on neural development, cell polarity, and signaling pathways .

CELSR1 antibodies are valuable tools for examining this protein's expression patterns, localization, and interactions within neural tissues. Biotin-conjugated variants offer enhanced detection sensitivity through the streptavidin-biotin interaction system.

What are the structural characteristics of biotin-conjugated CELSR1 antibodies?

Biotin-conjugated CELSR1 antibodies typically consist of:

• Primary antibody structure: Usually polyclonal or monoclonal IgG raised in rabbit against specific CELSR1 epitopes

• Conjugation chemistry: Biotin molecules covalently attached to the antibody, typically through amine/ester chemistry

• Antibody target: Specific regions of the CELSR1 protein, such as peptide sequences from positions 345-363 AA in human CELSR1

• Formulation: Commonly supplied in buffers containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (e.g., 50% Glycerol, 0.01M PBS, pH 7.4)

The biotin moiety provides a strong affinity binding site for streptavidin-conjugated detection reagents, enabling sensitive visualization or quantification in various experimental platforms .

How does biotin conjugation influence antibody performance?

Biotin conjugation affects antibody performance in several ways:

• Enhanced detection sensitivity through signal amplification, as multiple streptavidin molecules can bind to each biotinylated antibody

• Potential modification of antibody binding kinetics, particularly if biotin molecules are conjugated near the antigen-binding region

• Increased versatility in detection methods, as biotinylated antibodies can be detected using various streptavidin-conjugated reporter molecules

• Extended shelf-life compared to some directly labeled antibodies, though proper storage remains essential

• Possibility of interference in samples with high endogenous biotin content

Optimal performance requires careful consideration of the biotin-to-antibody ratio, which ideally ranges from 2-3 biotin molecules per antibody for most applications .

What experimental techniques are compatible with biotin-conjugated CELSR1 antibodies?

Biotin-conjugated CELSR1 antibodies are versatile reagents suitable for multiple techniques:

For sandwich ELISA applications specifically, using matched antibody pairs (e.g., CELSR1 capture and detector antibodies) can provide greater specificity and sensitivity for target detection .

How should CELSR1 Antibody, Biotin conjugated be stored and handled to maintain activity?

Proper storage and handling are critical for maintaining antibody functionality:

• Storage temperature: Store at -20°C or -80°C upon receipt

• Freeze/thaw cycles: Avoid repeated freezing and thawing; make small working aliquots

• Buffer conditions: Typically supplied in stabilizing buffers containing glycerol (50%) and preservatives

• Working dilutions: Prepare fresh and use within recommended time frames

• Temperature during use: Maintain at refrigerated temperatures when not in use during experimental procedures

• Contamination prevention: Use sterile technique when handling to prevent microbial contamination

Long-term storage stability can be enhanced by adding glycerol 1:1 before freezing if not already present in the formulation .

What considerations are important when designing a sandwich ELISA using biotin-conjugated CELSR1 antibodies?

Designing an effective sandwich ELISA with biotin-conjugated CELSR1 antibodies requires:

• Antibody pair selection: Use validated pairs with one capture antibody (typically at 2 μg/mL) and one detector antibody (typically at 0.5 μg/mL)

• Plate preparation: Coat high-binding ELISA plates with capture antibody in appropriate buffer

• Blocking optimization: Test different blocking agents to minimize background while maintaining specific signal

• Sample preparation: Determine appropriate dilutions and pre-treatments for your specific sample type

• Detection system: Select appropriate streptavidin-conjugated detection reagent (HRP is common)

• Standard curve: Include recombinant CELSR1 protein standards at known concentrations

• Controls: Include positive controls, negative controls, and background controls

• Validation: Assess specificity, sensitivity, reproducibility, and dynamic range

Optimization through checkerboard titration experiments helps determine the ideal concentrations of capture antibody, detector antibody, and detection reagent for maximum sensitivity and specificity .

How can researchers address problems with high background when using biotin-conjugated antibodies?

High background is a common challenge with biotin-conjugated antibodies that can be addressed through several strategies:

• Endogenous biotin blocking:

  • For tissue samples, pre-block with unconjugated avidin/streptavidin followed by free biotin

  • Use commercial biotin blocking kits for tissues known to contain high biotin levels

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time or concentration

  • Include detergents like Tween-20 at appropriate concentrations

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • More dilute antibody solutions often reduce non-specific binding

• Washing optimization:

  • Increase wash stringency (more washes, longer duration)

  • Use appropriate buffers with detergents

• Sample-specific strategies:

  • Pre-absorb antibodies with relevant tissues/proteins

  • Include tissue-specific blocking agents

Researchers should systematically test these approaches to determine which factors are contributing to background in their specific experimental system .

What methods can be used to validate the specificity of biotin-conjugated CELSR1 antibodies?

Comprehensive validation of antibody specificity includes:

• Protein-level validation:

  • Western blot analysis to confirm detection of protein with expected molecular weight

  • Immunoprecipitation followed by mass spectrometry identification

• Genetic validation:

  • Testing in CELSR1 knockout or knockdown models

  • Correlation of staining intensity with known expression levels

• Epitope validation:

  • Competition assays with immunizing peptide

  • Testing multiple antibodies against different CELSR1 epitopes

• Cross-reactivity assessment:

  • Testing against related proteins (other cadherins)

  • Testing in systems with controlled expression

• Control experiments:

  • Inclusion of isotype controls

  • Secondary-only controls to assess non-specific binding

These validation steps help ensure that observed signals truly represent CELSR1 rather than non-specific binding or cross-reactivity .

What factors influence the efficiency of biotin conjugation to antibodies?

The efficiency of biotin conjugation depends on several factors:

• Chemistry parameters:

  • pH of reaction (typically 7.2-8.5 for amine-directed conjugation)

  • Temperature (usually room temperature for 1-2 hours)

  • Molar ratio of biotin reagent to antibody (controls degree of labeling)

  • Reaction time

• Antibody considerations:

  • Purity of starting antibody (carrier proteins like BSA will compete for biotinylation)

  • Concentration (typically 1-10 mg/mL is optimal)

  • Buffer composition (avoid amine-containing buffers like Tris)

  • Antibody stability under reaction conditions

• Biotin reagent selection:

  • Water-soluble vs. organic-soluble reagents

  • Spacer arm length (longer spacers improve streptavidin binding)

  • Reactive group (NHS esters for amine coupling, maleimides for thiol coupling)

Commercial kits like BiotinPure™ typically optimize these parameters to achieve 2-3 biotins per antibody, which represents an optimal balance between conjugation efficiency and preserved antibody activity .

How should researchers design experiments to quantitatively evaluate CELSR1 expression?

Quantitative assessment of CELSR1 expression requires careful experimental design:

• Standard curve establishment:

  • Use recombinant CELSR1 protein at known concentrations

  • Include multiple replicates and assess inter-assay variability

• Sample preparation considerations:

  • Standardize cell/tissue lysis conditions

  • Normalize protein loading (total protein quantification)

  • Consider subcellular fractionation if studying localization

• Control selection:

  • Include positive controls (tissues/cells with known CELSR1 expression)

  • Include negative controls (tissues/cells with minimal CELSR1 expression)

  • Include technical controls (isotype, secondary-only)

• Quantification approach:

  • For ELISA: Calculate concentration based on standard curve

  • For immunoblotting: Densitometry normalized to loading controls

  • For imaging: Mean fluorescence intensity with background subtraction

• Statistical analysis:

  • Determine appropriate statistical tests based on experimental design

  • Account for technical and biological replicates

  • Report measures of central tendency and variation

This systematic approach helps ensure reproducible, quantitative assessment of CELSR1 expression levels .

What considerations are important for multiplex experiments incorporating biotin-conjugated CELSR1 antibodies?

Multiplex experimental design requires careful planning:

• Detection system compatibility:

  • Ensure spectral separation between fluorophores if using fluorescent detection

  • Consider signal strength differences between targets

• Antibody combinations:

  • Verify absence of cross-reactivity between antibodies

  • Consider using the biotin-streptavidin system for the least abundant target

  • Test each antibody individually before combining

• Protocol optimization:

  • Sequence of antibody application may be critical

  • May require separate incubation steps for different antibodies

  • Blocking conditions may need adjustment for multiple antibodies

• Controls for multiplex systems:

  • Single-stained controls for each target

  • Fluorescence minus one (FMO) controls

  • Isotype controls for each antibody species and class

• Analysis considerations:

  • Compensation for spectral overlap if using fluorescent detection

  • Potential need for linear unmixing algorithms

  • Sequential imaging may be preferable to simultaneous in some cases

These considerations help ensure reliable data interpretation in complex multiplex experimental systems .

How can researchers determine the dynamic range and sensitivity limits of biotin-conjugated CELSR1 antibody assays?

Characterizing assay performance parameters requires systematic evaluation:

• Standard curve analysis:

  • Create a dilution series of recombinant CELSR1 protein

  • Plot signal vs. concentration (log scale often appropriate)

  • Identify linear range and regions of non-linearity

• Sensitivity determination:

  • Calculate lower limit of detection (LOD): typically 3 standard deviations above background

  • Determine lower limit of quantification (LLOQ): typically 10 standard deviations above background

  • Assess functional sensitivity through precision profile analysis

• Dynamic range assessment:

  • Determine the range between LLOQ and the highest concentration with acceptable linearity

  • Express as orders of magnitude (e.g., 3-log dynamic range)

• Precision analysis:

  • Calculate intra-assay CV% (multiple replicates in same experiment)

  • Calculate inter-assay CV% (same samples across multiple experiments)

  • Evaluate precision across the working range

• Recovery and parallelism testing:

  • Spike known amounts of recombinant protein into sample matrix

  • Analyze dilution linearity of endogenous samples

These parameters provide critical information for experimental planning and interpretation of quantitative data .

How can biotin-conjugated CELSR1 antibodies be integrated into advanced imaging techniques?

Biotin-conjugated CELSR1 antibodies can enhance advanced imaging through:

• Super-resolution microscopy:

  • Use with streptavidin-conjugated fluorophores designed for STORM, PALM, or STED

  • The biotin-streptavidin system provides amplification beneficial for single-molecule localization

  • Can be combined with click chemistry approaches for multiplexing

• Expansion microscopy:

  • Biotin-streptavidin linkages offer strong binding during sample processing

  • Streptavidin-fluorophore conjugates can be applied post-expansion

• Correlative light and electron microscopy (CLEM):

  • Biotin can be detected with streptavidin-gold for electron microscopy

  • Same sample can be processed for both fluorescence and EM imaging

• Intravital imaging applications:

  • Biotin-conjugated antibody fragments may be used for in vivo imaging

  • Can be paired with near-infrared fluorophores for deep tissue penetration

These advanced applications capitalize on the strong biotin-streptavidin interaction combined with the specificity of CELSR1 antibodies .

What approaches can address anamnestic antibody responses in longitudinal studies using biotin-labeled reagents?

Research involving repeated exposure to biotinylated reagents must consider immune responses:

• Recognition of potential immune reactions:

  • Antibodies against biotin-labeled components can develop upon re-exposure

  • These may be primarily IgG1 subclass with specificity for the biotin epitope

• Experimental design strategies:

  • Include controls to assess anti-biotin antibody development

  • Consider preliminary screening of subjects for pre-existing anti-biotin antibodies

  • Plan for potential accelerated clearance of biotin-labeled components in follow-up studies

• Alternative approaches:

  • Use different conjugation chemistries for follow-up studies

  • Consider alternative detection systems in subjects with known anti-biotin responses

  • Implement epitope masking strategies if biotin conjugates must be used

• Monitoring recommendations:

  • Test for binding of human anti-IgG to biotin-labeled components

  • Assess changes in clearance kinetics of biotin-labeled reagents

  • Monitor for clinical or laboratory adverse events, though these are typically not observed

These considerations are particularly important for longitudinal studies involving repeated administration of biotin-labeled reagents .