DYM Antibody, Biotin conjugated

What is DYM Antibody and what is its target protein?

DYM Antibody targets Dymeclin (also known as Dyggve-Melchior-Clausen syndrome protein), which is encoded by the DYM gene with UniProtID Q7RTS9. The specific biotin-conjugated version is a polyclonal antibody raised in rabbits using recombinant Human Dymeclin protein (amino acids 224-348) as the immunogen . This antibody is designed for research applications in signal transduction pathways and is purified using Protein G methodology with >95% purity . Dymeclin plays important roles in cellular trafficking and Golgi apparatus organization, making it relevant for studies investigating intracellular transport mechanisms.

What are the benefits of using biotin-conjugated antibodies in protein detection systems?

Biotin-conjugated antibodies provide significant advantages for protein detection, particularly for low-abundance targets like Dymeclin. The biotin-streptavidin/avidin system creates a powerful signal amplification cascade due to:

• Exceptionally strong non-covalent interactions between biotin and streptavidin/avidin

• The ability of each streptavidin/avidin molecule to bind up to four biotin molecules, creating large molecular complexes

• Enhanced sensitivity compared to direct detection methods

• Versatility in detection systems, as biotinylated antibodies can be coupled with various reporter molecules

This signal amplification capability is critical for detecting proteins expressed at low levels, providing sensitivity that would be unattainable with conventional direct detection methods .

What experimental applications are validated for DYM Antibody, Biotin conjugated?

The primary validated application for DYM Antibody, Biotin conjugated is ELISA (Enzyme-Linked Immunosorbent Assay) . This antibody has been specifically optimized for:

• Detection of human Dymeclin protein in ELISA formats

• Signal transduction research applications

• Potential compatibility with immunohistochemistry and immunocytochemistry when using appropriate signal amplification techniques

The biotin conjugation makes this antibody particularly versatile for experimental protocols requiring signal enhancement, such as detection of low-abundance Dymeclin protein in complex biological samples .

How does the storage buffer composition affect DYM Antibody stability and performance?

The DYM Antibody, Biotin conjugated is supplied in a specialized buffer formulation consisting of:

This buffer composition is specifically designed to maintain antibody stability during storage while preserving both the immunoreactivity of the antibody and the integrity of the biotin conjugation . The high glycerol content allows for storage at -20°C without complete freezing, which minimizes damage from ice crystal formation. Researchers should note that this buffer composition must be considered when calculating working dilutions for experimental applications.

What are the critical differences between ABC and LSAB methods when using biotinylated DYM antibody?

When designing experiments with biotinylated DYM antibody, researchers should consider the distinct characteristics of ABC (Avidin-Biotin Complex) and LSAB (Labeled Streptavidin-Biotin) methods:

The LSAB method has become increasingly favored for detecting proteins like Dymeclin due to its improved specificity and simplified protocol. This method is particularly advantageous when working with tissue samples or when background signal is a significant concern .

How can researchers optimize detection of low-abundance Dymeclin protein using biotinylated antibodies?

Detecting low-abundance Dymeclin protein requires careful optimization of several experimental parameters:

• Signal amplification strategy selection:

  • LSAB typically provides better signal-to-noise ratio than ABC for low-abundance targets

  • Consider multi-layer amplification for extremely low expression levels

• Background reduction techniques:

  • Implement comprehensive blocking protocols (protein blockers plus avidin/biotin blocking)

  • Consider anti-biotin antibody approaches when avidin/streptavidin produces high background

  • Optimize washing buffers with appropriate detergent concentrations

• Antibody titration:

  • Perform systematic dilution series to determine optimal concentration

  • Too high concentration increases background while too low reduces specific signal

• Detection reagent selection:

  • Avoid blue fluorescent dyes (e.g., CF®405S) for low-abundance targets due to their lower fluorescence intensity and higher background characteristics

  • Consider enhanced chemiluminescent substrates for Western blotting applications

• Incubation optimization:

  • Extended incubation times (overnight at 4°C) often improve sensitivity

  • Temperature control is critical for reproducible results

These optimizations collectively enhance the detection of low-abundance Dymeclin while minimizing background interference that could obscure genuine signals.

How can I validate the specificity of DYM Antibody, Biotin conjugated in my experimental system?

Comprehensive validation of DYM Antibody specificity requires a multi-faceted approach:

• Expression correlation analysis:

  • Compare protein detection with known mRNA expression patterns

  • Utilize tissues/cells with documented Dymeclin expression levels

  • Correlation between detection signal and expected expression provides evidence of specificity

• Knockdown/knockout validation:

  • Test antibody in cells with DYM gene knockdown/knockout

  • Significant signal reduction in knockdown samples supports specificity

  • Complete signal loss in knockout samples provides strong evidence of specificity

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide (amino acids 224-348 of Dymeclin)

  • Specific binding should be blocked by peptide competition

• Cross-reactivity assessment:

  • Test antibody in samples from different species (antibody is designed for human reactivity)

  • Evaluate potential cross-reactivity with structurally similar proteins

• Alternative antibody comparison:

  • Compare detection patterns with other antibodies targeting different Dymeclin epitopes

  • Concordant results from multiple antibodies increase confidence in specificity

• Recombinant protein controls:

  • Use purified recombinant Dymeclin as positive control

  • Validate detection of the specific 224-348AA region used as immunogen

How can I combine MACS and surface biotinylation when working with DYM Antibody for cell-specific protein analysis?

Integrating Magnetically Activated Cell Sorting (MACS) with DYM Antibody biotinylation enables sophisticated cell-specific protein analysis through this methodological workflow:

• Cell preparation and MACS labeling:

  • Prepare single-cell suspensions with >90% viability

  • Label target cell population with appropriate MACS microbeads

  • Load labeled cells onto MACS column within a magnetic field

• In-column biotinylation protocol:

  • While target cells remain magnetized in column, prepare fresh biotinylation reagent

  • Dissolve biotin in a nitrogen-filled chamber to maintain optimal reactivity

  • Pass biotin solution through the column containing retained cells

  • This approach minimizes membrane disruption during biotinylation

• Elution and validation:

  • Remove column from magnetic field and elute cells

  • Collect approximately 10^6 cells (75 μL) to validate biotinylation success

  • Perform flow cytometry validation using streptavidin-FITC (3 μL in 100 μL cell suspension)

  • Include 7-AAD staining to assess membrane integrity

• Protein isolation and analysis:

  • Lyse remaining cells in appropriate buffer

  • Capture biotinylated proteins using streptavidin beads

  • Process for downstream applications (Western blot, mass spectrometry)

This integrated approach enables precise analysis of the Dymeclin protein in specific cell subsets, providing insights into cell type-specific expression and interactions.

What are the critical parameters for optimizing DYM Antibody, Biotin conjugated in ELISA protocols?

Optimization of ELISA protocols using DYM Antibody, Biotin conjugated requires careful attention to several critical parameters:

• Antibody concentration optimization:

  • Perform systematic titration (typically 0.1-10 μg/mL range)

  • Evaluate signal-to-noise ratio at each concentration

  • Select concentration that maximizes specific signal while minimizing background

• Detection system selection:

  • Streptavidin-HRP is commonly used for colorimetric detection

  • Streptavidin-poly-HRP provides enhanced sensitivity

  • Fluorescent detection systems may offer improved dynamic range

• Incubation conditions:

  • Temperature: Room temperature vs. 4°C (longer incubations often benefit from 4°C)

  • Duration: 1-2 hours standard, overnight for maximum sensitivity

  • Agitation: Gentle orbital shaking improves binding kinetics and uniformity

• Buffer optimization:

  • Blocking buffer: BSA vs. casein-based blockers (casein often provides lower background)

  • Diluent composition: Match antibody buffer (0.01M PBS, pH 7.4) for stability

  • Wash buffer: PBS-Tween concentration affects stringency

• Plate selection:

  • High-binding polystyrene plates for direct coating

  • NeutrAvidin/streptavidin-coated plates for capture assays

  • Black plates for fluorescent detection, clear for colorimetric

• Sample preparation:

  • Pre-clearing steps to remove potentially interfering components

  • Dilution series to ensure measurements within linear range

  • Addition of protease inhibitors for sensitive samples

Systematic optimization of these parameters will establish a robust ELISA protocol for specific detection of Dymeclin protein in experimental samples.

What protocol modifications are required when using DYM Antibody, Biotin conjugated in flow cytometry applications?

Adapting DYM Antibody, Biotin conjugated for flow cytometry applications requires specific protocol modifications:

• Cell preparation:

  • For intracellular Dymeclin detection, use appropriate fixation/permeabilization reagents

  • Maintain high cell viability prior to fixation

  • Ensure single-cell suspensions to prevent aggregates

• Staining protocol adjustments:

  • Primary staining: Incubate with DYM Antibody, Biotin conjugated (typically 0.5-5 μg/mL)

  • Secondary detection: Use fluorophore-conjugated streptavidin

  • Select streptavidin-fluorophore conjugates compatible with your cytometer configuration

  • Avoid blue fluorescent dyes (e.g., CF®405S) for low-abundance targets due to higher background

• Essential controls:

  • Unstained cells for autofluorescence baseline

  • Isotype control (biotin-conjugated rabbit IgG)

  • Secondary-only control (streptavidin-fluorophore without primary antibody)

  • Biological negative controls (cells not expressing Dymeclin)

  • 7-AAD or alternative viability dye to exclude dead cells

• Instrument settings:

  • Optimize PMT voltages using unstained and single-stained controls

  • Set compensation if using multiple fluorophores

  • Use appropriate threshold settings to exclude debris

• Data analysis considerations:

  • Gate on viable single cells only

  • Compare signal to appropriate negative controls

  • Consider median fluorescence intensity rather than mean for non-normally distributed signals

These modifications will enable accurate detection and quantification of Dymeclin protein in heterogeneous cell populations using flow cytometry.

What troubleshooting strategies should be employed for non-specific binding issues with biotinylated DYM antibody?

When encountering non-specific binding with DYM Antibody, Biotin conjugated, implement these systematic troubleshooting strategies:

• Endogenous biotin blocking:

  • Biological samples often contain endogenous biotin

  • Implement avidin/biotin blocking kit prior to adding biotinylated antibodies

  • Incubate with free avidin followed by free biotin to block endogenous biotin

• Detection system optimization:

  • Compare LSAB vs. ABC methods (LSAB typically produces lower background)

  • Consider using anti-biotin antibody instead of direct avidin/streptavidin detection

  • Titrate streptavidin-conjugate concentration to minimize non-specific binding

• Buffer modifications:

  • Increase blocking protein concentration (typically 2-5%)

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

  • Include 0.1-0.5M NaCl to reduce ionic interactions

  • Add 1-5% serum matched to host species of secondary reagent

• Antibody dilution optimization:

  • Excessive antibody concentration is a common cause of non-specific binding

  • Perform systematic dilution series (typically 1:100 to 1:10,000)

  • Select concentration that optimizes signal-to-noise ratio

• Washing protocol enhancement:

  • Increase wash buffer volume (at least 3-5× reaction volume)

  • Extend washing times (3-5 minutes per wash)

  • Increase number of wash steps (minimum 3-5 washes)

  • Ensure complete removal of wash buffer between steps

• Sample-specific interventions:

  • For tissue sections: additional peroxidase/phosphatase blocking

  • For cell lysates: pre-clearing with protein A/G or non-immune IgG

  • For recombinant proteins: additional purification steps

Systematic application of these strategies will help identify and resolve the specific source of non-specific binding in your experimental system.

What is the optimal workflow for combining DYM Antibody detection with mass spectrometry analysis?

Integrating DYM Antibody labeling with mass spectrometry requires a carefully optimized workflow:

• Sample preparation:

  • For cell surface protein analysis, implement in-column biotinylation if using MACS

  • For intracellular proteins, use cell-permeable biotinylation reagents

  • Validate biotinylation success using flow cytometry with streptavidin-FITC detection

• Cell lysis and protein extraction:

  • Use MS-compatible lysis buffers (avoid detergents that interfere with MS)

  • Include protease and phosphatase inhibitors to preserve protein modifications

  • Perform lysis at 4°C to minimize proteolysis

• Enrichment of biotinylated proteins:

  • Capture biotinylated proteins using high-capacity streptavidin beads

  • Wash extensively to remove non-specifically bound proteins

  • Consider on-bead digestion to minimize sample loss

• Sample preparation for MS:

  • Perform reduction with Tris(2-carboxyethyl)phosphine (TCEP) in 50 mM TEAB

  • Alkylate with Iodoacetamide (IAA) in 50 mM TEAB

  • Digest with high-quality trypsin in 0.2% RapiGest-TEAB solution

• Peptide purification:

  • Use in-stage tip purification methodology

  • Condition with 0.1% TFA and 50% ACN

  • Wash with 0.1% TFA

  • Elute with 0.1% TFA and 80% ACN

• MS analysis parameters:

  • Consider using data-dependent acquisition for discovery

  • Implement parallel reaction monitoring for targeted analysis

  • Include biotin modifications in search parameters

• Data analysis approach:

  • Filter against appropriate controls (non-biotinylated samples)

  • Apply stringent false discovery rate thresholds

  • Validate key findings with orthogonal techniques

This workflow enables comprehensive proteomic analysis of Dymeclin interactions and modifications, providing deeper insights into its biological functions.

What validation controls are essential when using DYM Antibody, Biotin conjugated in multiplex protein detection systems?

Robust validation of multiplex assays using DYM Antibody, Biotin conjugated requires a comprehensive set of controls:

• Specificity controls:

  • Peptide competition: Pre-incubate antibody with immunizing peptide (Dymeclin 224-348AA)

  • Genetic validation: Test antibody in DYM knockdown/knockout samples

  • Cross-reactivity assessment: Test with structurally similar proteins

• Technical controls:

  • Single-stained controls for each detection reagent

  • Fluorescence minus one (FMO) controls for multiplex flow cytometry

  • Biotin blocking controls to assess endogenous biotin interference

  • Isotype control (biotin-conjugated rabbit IgG)

  • Secondary-only control (detection reagent without primary antibody)

• Sample quality controls:

  • Viability assessment (7-AAD staining) for cellular applications

  • Sample processing controls that undergo all steps except antibody addition

  • Time-course stability controls to assess sample degradation effects

• Quantification controls:

  • Standard curves using recombinant Dymeclin protein

  • Inter-assay calibrators to normalize between experimental runs

  • Spike-in controls at known concentrations for recovery assessment

• System-specific controls:

  • For multiplexed detection: assess antibody cross-reactivity in the multiplex format

  • For microscopy: auto-fluorescence controls and spectral unmixing controls

  • For mass spectrometry: isotope-labeled internal standards

Implementing these validation controls ensures reliable data interpretation and facilitates troubleshooting of any anomalous results in multiplex detection systems.