CHRND Antibody, FITC conjugated

What is CHRND and why are FITC-conjugated antibodies used to study it?

CHRND (Cholinergic Receptor, Nicotinic, delta subunit) is one of the five subunits comprising the muscle acetylcholine receptor (AChR). After acetylcholine binding, the AChR undergoes an extensive conformational change affecting all subunits, leading to the opening of an ion-conducting channel across the plasma membrane .

FITC-conjugated CHRND antibodies enable direct visualization of this receptor subunit without requiring secondary detection steps. The FITC conjugate has an excitation/emission profile of approximately 499/515 nm, making it compatible with standard 488 nm laser lines in flow cytometry and fluorescence microscopy . This direct labeling approach offers several advantages:

• Direct visualization in live or fixed cells

• Capability for multiplexing with other fluorophores

• Quantitative analysis of receptor expression

• Elimination of secondary antibody incubation steps

• Reduced background compared to indirect methods

What are the typical applications for CHRND Antibody, FITC conjugated?

CHRND Antibody, FITC conjugated serves diverse research applications across neuroscience and immunology fields:

In myasthenia gravis research, fluorescently-labeled antibodies are particularly valuable for cell-based assays that detect autoantibodies against clustered AChR. The KL525 score (percentage of FITC-positive cells) provides a quantitative measure of anti-AChR antibodies in patient serum with significantly higher sensitivity than traditional methods .

How should CHRND Antibody, FITC conjugated be stored for optimal performance?

Proper storage is critical for maintaining FITC conjugate integrity and antibody function:

Research indicates that repeated freeze-thaw cycles significantly reduce both antibody binding efficiency and FITC fluorescence intensity, making proper aliquoting essential for experimental reproducibility .

What dilutions are recommended for different experimental techniques?

Based on published protocols and manufacturer recommendations:

It's important to note that "optimal dilutions/concentrations should be determined by the end user" as stated in multiple product datasheets, emphasizing the importance of antibody titration experiments for each specific application and experimental system .

How can CHRND Antibody, FITC conjugated be used in flow cytometry for diagnosing myasthenia gravis?

Flow cytometry with FITC-conjugated CHRND antibodies provides a powerful diagnostic approach for myasthenia gravis (MG), particularly for detecting anti-AChR antibodies in patients previously considered seronegative by traditional methods:

Protocol Framework:

• Cell Preparation:

  • Culture cells expressing clustered AChR (such as the KL525 stable cell line) at approximately 10^6 cells/well

  • Harvest cells by centrifugation and wash 3× with PBS

  • Resuspend cells in 100μl PBS for antibody labeling

• Antibody Incubation:

  • Incubate cells with patient serum (1:200 dilution) and an AChR δ antibody (1:200)

  • For detection, employ 488-labeled anti-human secondary antibody (1:300) and APC-labeled anti-mouse secondary antibody (1:500)

• Flow Cytometry Analysis:

  • Analyze immediately using a flow cytometer with appropriate lasers (488nm for FITC)

  • Calculate the KL525 score as the percentage of double-positive cells

  • Apply threshold determination (mean + 2 SDs of negative controls)

A key advantage of this cell-based assay approach is its ability to detect low-affinity clustered anti-AChR antibodies missed by radioimmunoprecipitation assay (RIPA). Research demonstrates that "the positive results of MG patients reported by the KL525 was 80.6% (83/103), 29.1% higher than the 51.4% (53/103) of RIPA" .

How can specificity of CHRND Antibody, FITC conjugated be verified in experimental settings?

Rigorous validation of CHRND antibody specificity is essential for reliable research outcomes. A comprehensive validation strategy includes:

Multiple Control Systems:

• Positive Controls: Use cell lines with confirmed CHRND expression or recombinant protein

• Negative Controls: Include cell lines lacking CHRND expression, isotype controls, and secondary-only controls

• Genetic Controls: Compare results between wild-type and CHRND-knockdown/knockout models

Technical Validation Approaches:

When performing flow cytometry validation, researchers should compare staining profiles between CHRND-positive and negative populations and analyze the shift in fluorescence intensity relative to unstained controls .

What are the technical challenges in distinguishing between clustered versus non-clustered AChR using CHRND Antibody, FITC conjugated?

Differentiating between clustered and non-clustered AChRs presents significant technical challenges, requiring sophisticated methodological approaches:

Biological Significance:
In neuromuscular junctions, AChRs exist as pentamers consisting of α, β, γ, and δ subunits clustered in their native conformational state . This clustering is physiologically relevant, as "clustered anti-AChR antibodies with low affinity" are present in seronegative myasthenia gravis patients but cannot be detected using traditional methods that employ solubilized receptors .

Key Technical Challenges:

• Spatial Resolution Requirements:

  • AChR clusters may be submicron in size, requiring super-resolution microscopy techniques

  • Conventional microscopy may not distinguish between closely spaced individual receptors

• Antibody-Induced Clustering:

  • The antibodies themselves can induce artificial clustering: "We used antibody-mediated cross-linking of CHRN to induce receptor endocytosis in C2C12 muscle cells"

  • Solution: Use monovalent antibody fragments (Fab) or fixed samples where clustering is preserved before antibody application

• Verification of Biological Clustering:

  • Co-visualization of clustering proteins like rapsyn is essential

  • "By coexpressing AChR subunits on the plasma membrane with rapsyn, a synaptic protein that induces AChR clustering, cell-based assays offer a highly natural membrane environment"

Methodological Solutions:

Research on the stable KL525 cell line expressing clustered AChR has demonstrated that properly clustered receptors are essential for detecting clinically relevant antibodies in myasthenia gravis patients, highlighting the importance of distinguishing between clustered and non-clustered receptor populations .

How can CHRND Antibody, FITC conjugated be used to study acetylcholine receptor endocytosis?

FITC-conjugated CHRND antibodies enable real-time visualization of AChR trafficking and internalization dynamics:

Experimental Protocol:

• Surface Receptor Labeling:

  • Plate muscle cells (e.g., C2C12) on imaging-compatible surfaces

  • Incubate with FITC-conjugated CHRND antibody at 4°C (endocytosis-inhibiting condition)

  • Wash thoroughly to remove unbound antibody

• Endocytosis Induction and Monitoring:

  • Shift cells to 37°C to permit endocytosis

  • Collect cells at defined time points (0, 5, 15, 30, 60 minutes)

  • "After incubating with Alexa Fluor 488-conjugated antibody," researchers can track the internalization process

• Surface vs. Internalized Receptor Distinction:

  • Acid Wash Method: Brief treatment with pH 2.5-3.0 buffer to strip remaining surface antibodies

  • Differential Labeling: Use a spectrally distinct secondary antibody to label remaining surface receptors

• Mechanistic Analysis:

  • Pretreat cells with endocytic pathway inhibitors to identify internalization mechanisms

  • Perform co-localization studies with endosomal markers

  • Investigate protein interactions: "CHRN interacted with BAG3 and HSPA8" suggests involvement of the CASA complex in receptor processing

This approach allows detailed characterization of receptor endocytosis dynamics, which is particularly relevant for understanding receptor downregulation in pathological conditions like myasthenia gravis.

What are the considerations for using CHRND Antibody, FITC conjugated in multiplex imaging?

Successful multiplex imaging with FITC-conjugated CHRND antibodies requires careful experimental design:

Spectral Considerations:

Practical Implementation:
Research examples show successful multiplex applications where "CHRND staining (green) combined with F-Actin staining using Phalloidin (red) and nuclei with DAPI (blue)" provided clear distinction between cellular components.

Technical Optimization Strategies:

• Signal Balancing:

  • Adjust antibody concentrations and exposure settings for each channel

  • FITC brightness is moderate compared to newer fluorophores, so signal optimization is crucial

• Photobleaching Management:

  • FITC is relatively susceptible to photobleaching

  • Use anti-fade mounting media and minimize exposure times

  • Acquire FITC channel images first in sequential imaging workflows

• Cross-Talk Minimization:

  • Employ sequential acquisition when spectral overlap exists

  • Perform proper compensation using single-stained controls

  • Use spectral unmixing algorithms for overlapping fluorophores

The implementation of these strategies ensures accurate discrimination between signals in multiplexed experiments, allowing researchers to simultaneously visualize CHRND alongside other cellular markers.