CHRND Antibody, HRP conjugated

What Is CHRND Antibody, HRP Conjugated and What Are Its Primary Research Applications?

CHRND Antibody, HRP conjugated is a specialized research tool targeting the delta subunit of the nicotinic acetylcholine receptor with horseradish peroxidase directly attached to the antibody molecule. This combination enables:

• Direct detection in immunoassays without requiring secondary antibodies

• Primary applications: Western blotting, ELISA, and immunohistochemistry (IHC)

• Research focus areas: Neuromuscular junction disorders, congenital myasthenic syndromes, and neuroscience research

CHRND antibodies can detect both human and other mammalian CHRND proteins depending on the specific antibody clone. For example, certain antibodies like clone C-4 can detect CHRND from mouse, rat, and human origin across multiple applications .

How Does HRP Conjugation Chemistry Affect Antibody Performance?

The conjugation of HRP to CHRND antibodies involves specific chemical processes that researchers should understand:

• Molecular weight impact: HRP (44 kDa) adds substantial mass to antibodies (150 kDa IgG), creating conjugates of approximately 238-326 kDa depending on the HRP:antibody ratio

• Conjugation ratio: Typically 2-4 HRP molecules per antibody molecule, determined by A403 and A280 readings

• Buffer considerations: Common buffer additives can inhibit conjugation efficiency

Avoid buffers containing thiomersal/thimerosal, merthioloate, sodium azide, glycine, proclin, and nucleophilic components (primary amines and thiols) .

What Are the Optimal Dilution Factors for Different Experimental Applications?

Application-specific dilution factors significantly impact experimental success:

For CHRND Antibody (88B):

Application notes: This antibody detects approximately 60 kDa protein (gamma subunit) and 65 kDa protein (delta subunit) from torpedo skeletal muscle homogenates. Under non-reducing conditions, the delta subunit migrates mostly as a dimer of ~130 kDa .

For other CHRND antibodies, optimal dilution factors may vary significantly, so pilot experiments are recommended for each new antibody and application.

How Should Researchers Optimize Detection Systems for CHRND Antibody, HRP Conjugated?

Optimizing chromogenic/chemiluminescent substrates is crucial for maximum sensitivity:

• Chromogenic substrates:

  • Diaminobenzidine (DAB) produces water-insoluble brown precipitate

  • ABTS produces soluble green product

  • TMB (3,3',5,5'-Tetramethylbenzidine) produces blue product transitioning to yellow with stop solution

• Protocol optimization steps:

  • Perform antibody titration experiments

  • Optimize substrate incubation time (typically 15 minutes for TMB)

  • Read optical density at appropriate wavelength (450nm for TMB after stop solution addition)

  • Implement proper washing steps to reduce background (PBS containing 0.05% Tween 20)

For enhanced sensitivity in detection of low-abundance targets, extended substrate incubation times may be necessary, with careful monitoring to prevent signal saturation.

What Validation Methods Should Be Used to Confirm CHRND Antibody Specificity?

Comprehensive validation is essential for reliable research outcomes:

• Control experiments:

  • Positive controls: Torpedo skeletal muscle homogenates or transfected cells expressing CHRND

  • Negative controls: Non-transfected cells or tissues known not to express CHRND

  • Peptide competition assays: Pre-incubation with immunizing peptide should abolish specific signals

• Cross-reactivity assessment:

  • Test across multiple species if cross-reactivity is claimed (human, rat, mouse, etc.)

  • Verify specificity with knockout/knockdown models where available

  • Compare molecular weight of detected bands with predicted size (approximately 60-65 kDa)

In transfection studies, researchers successfully validated CHRND antibodies by demonstrating colocalization patterns in cells co-transfected with human AChR and rapsyn. Wild-type rapsyn resulted in colocalization of rapsyn and AChR in small dense clusters on the cell surface, providing a reliable validation system .

How Do CHRND Mutations Impact Antibody Recognition and Experimental Design?

Research involving CHRND mutations requires special considerations:

• Epitope location: Verify that the antibody's epitope region is not affected by the mutation of interest. For example, antibodies targeting AA 175-204 of CHRND may not detect mutations in that region

• Mutation-specific considerations:

  • E381K mutation in the long cytoplasmic loop affects AChR-rapsyn co-clustering

  • Microdeletions (such as the 2.2 kb deletion) disrupting the CHRND gene may result in no protein expression

  • Multiple congenital myasthenic syndrome phenotypes result from different CHRND mutation types

• Experimental adaptations:

  • For suspected mutation carriers, use antibodies targeting conserved regions

  • Include appropriate mutation-carrying cell lines as controls

  • Consider using multiple antibodies targeting different CHRND epitopes

The 2006 study by Müller et al. demonstrated how a specific CHRND E381K mutation severely reduced cluster formation compared to wild-type receptor, which has significant implications for antibody detection methods in mutation studies .

What Are the Critical Factors in Designing ELISA Protocols with CHRND Antibody, HRP Conjugated?

ELISA protocols require careful optimization for CHRND detection:

• Coating procedure:

  • Coat plates with recombinant AChRs (1 μg/ml) in 0.1M carbonate/bicarbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

  • Block with ELISA diluent (PBS containing 0.05M Tris-HCl, 0.2% casein, and 0.05% Tween 20) for 30 minutes at room temperature

• Antibody incubation:

  • Add serum samples at 1:100 dilution

  • Incubate for 2 hours at room temperature

  • If using unconjugated primary antibody, add HRP-conjugated secondary antibody (1:2,000 to 1:5,000)

  • Incubate for 90 minutes at room temperature

• Development and reading:

  • Add TMB solution and incubate for 15 minutes at room temperature

  • Add 2M H₂SO₄ as stop solution

  • Read optical density at 450nm within 30 minutes

For quantitative analysis, always include a standard curve using purified CHRND protein at known concentrations.

How Does Sample Preparation Influence CHRND Detection in Immunohistochemistry?

Sample preparation significantly impacts CHRND detection in tissue sections:

• Fixation considerations:

  • Formalin fixation may mask epitopes; use formaldehyde for cell cultures (as demonstrated in U251 and HeLa cells)

  • For tissue sections, cryostat preparation is recommended due to reactivity with the cytoplasmic side of the receptor

• Permeabilization requirements:

  • Required for accessing intracellular epitopes

  • Recommended for CHRND antibodies recognizing cytoplasmic domains

• Optimization protocol:

  • Fix cells with formaldehyde prior to staining

  • Probe with antibody at appropriate dilution (1:20-1:100) overnight at 4°C

  • Wash with PBS and incubate with fluorescent-conjugated secondary antibody (if not using direct HRP conjugate)

  • For visualization of cellular context, counterstain F-Actin with Phalloidin (red) and nuclei with DAPI (blue)

IHC staining of CHRND in rat skeletal muscle shows strong staining of the motor endplate, providing a useful positive control tissue .

What Strategies Can Address Non-specific Binding When Using CHRND Antibody, HRP Conjugated?

Non-specific binding can compromise experimental results:

• Blocking optimization:

  • For Western blots: 5% non-fat dry milk or 3-5% BSA in TBST

  • For IHC/ICC: 5% normal serum from the same species as the secondary antibody

  • For ELISA: PBS containing 0.05M Tris-HCl, 0.2% casein, and 0.05% Tween 20

• Washing protocol enhancement:

  • Increase number of wash steps (minimum 3×5 minutes)

  • Use appropriate detergent concentration (0.05-0.1% Tween 20)

  • Consider higher salt concentration in wash buffer for high background

• Antibody dilution optimization:

  • Perform titration experiments starting with manufacturer's recommended dilution

  • Increase dilution if background is excessive

  • Balance signal-to-noise ratio with detection sensitivity

For particularly challenging samples, consider pre-adsorption of the antibody with tissue/cell lysates from species with high cross-reactivity potential.

How Can CHRND Antibody, HRP Conjugated Be Applied in Research on Neuromuscular Junction Disorders?

CHRND antibodies are valuable tools in neuromuscular disease research:

• Congenital myasthenic syndromes (CMS):

  • Detect CHRND protein expression levels in patient samples

  • Study impact of mutations on receptor clustering and localization

  • Mutations in CHRND are associated with severe CMS phenotypes, often with early onset

• Multiple pterygium syndrome:

  • CHRND mutations can cause lethal multiple pterygium syndrome (LMPS)

  • HRP-conjugated antibodies can help analyze CHRND expression patterns during development

• Research methodologies:

  • Immunofluorescence co-localization studies with rapsyn to assess AChR clustering

  • Western blot analysis of protein expression levels

  • ELISA quantification of AChR antibodies in myasthenia gravis research

Studies have shown that CHRND mutations, such as the E381K mutation, can disrupt AChR-rapsyn co-clustering, demonstrating the importance of this specific interaction in neuromuscular junction formation and maintenance .