MYH4 Antibody, Biotin conjugated
Description
Introduction to MYH4 Antibody, Biotin Conjugated
The MYH4 Antibody, Biotin conjugated is a specialized immunological reagent designed for the detection and quantification of Myosin Heavy Chain 4 (MYH4), a skeletal muscle protein critical for muscle contraction . Biotin conjugation enables its use in biotin-avidin-based detection systems, such as enzyme-linked immunosorbent assays (ELISA), enhancing sensitivity and specificity .
Key Properties
Mechanism: The antibody binds MYH4 via its epitope, and biotin allows interaction with streptavidin-conjugated enzymes (e.g., HRP) for signal amplification in assays .
ELISA Performance
Data derived from MYH4 ELISA kits using biotin-conjugated antibodies demonstrate robust analytical performance :
| Parameter | Value |
|---|---|
| Detection Range | 0.32–20 ng/mL |
| Sensitivity | Minimum detectable dose (MDD): <0.156 ng/mL |
| Cross-reactivity | High specificity; no significant interference with analogues reported |
| Intra-assay Precision | CV <10% (20 replicates/plate) |
| Inter-assay Precision | CV <15% (3 plates, 20 replicates each) |
| Recovery | 83–106% in serum and cell culture media (spiked samples) |
Linearity: Serial dilutions (1:2 to 1:16) show average recovery rates of 82–101%, confirming proportional detection across concentrations .
Primary Use Cases
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Quantitative MYH4 Detection:
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Limitations:
Key Differences
Example:
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Unconjugated MYH4 Antibody (Proteintech): Validated for WB, IHC, IF; detects MYH4 in human, mouse, rat .
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FITC-Linked MYH4 Antibody (MyBioSource): Used in immunocytochemistry but lacks ELISA compatibility .
MYH4 Biology and Antibody Utility
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Role of MYH4:
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Antibody Applications in Studies:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Target Function: Muscle contraction.
Gene References and Function:
- MyHC-2b demonstrated consistent co-expression with all major MyHC isoforms, including both fast isoforms (MyHC-2a and -2x) and the slow isoform (MyHC-1). PMID: 23796659
Q&A
How does the immunogen region impact MYH4 antibody specificity in skeletal muscle studies?
The immunogen design determines epitope accessibility across MYH4 isoforms. For example:
Antibodies targeting the C-terminal rod domain (e.g., BioCat, Nordic Biosite) are optimal for detecting full-length MYH4 in Western blot (WB), as this region remains exposed in denatured proteins . Conversely, antibodies against conformational epitopes may fail in WB but excel in immunohistochemistry (IHC).
What validation criteria ensure MYH4 antibody specificity in murine models?
Three-tier validation is essential:
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Knockout Controls: Compare staining in wild-type vs. MYH4<sup>-/-</sup> tissues .
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Peptide Blocking: Pre-incubate antibody with immunogen peptide (10:1 molar ratio) to confirm signal loss .
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Cross-Reactivity Profiling: Test against recombinant MYH1, MYH2, and MYH7 isoforms to rule out off-target binding .
The ariel mouse model (MYH4 L342Q mutation) demonstrates the importance of isoform-specific validation, as aggregates containing mutant MYH4 may alter antibody accessibility .
How to optimize MYH4 antibody dilution for co-localization studies?
Empirical titration is critical due to tissue-specific epitope density:
| Application | Starting Dilution | Adjustments for Co-Localization |
|---|---|---|
| WB | 1:500 | Combine with streptavidin-HRP (1:10,000) |
| IHC | 1:50 | Reduce biotinylated antibody to 1:100 if background exceeds 5% signal/noise ratio |
For dual staining with α-actinin or titin antibodies, sequential labeling (MYH4 first, then non-biotinylated probes) minimizes steric interference .
How to resolve discrepancies in MYH4 expression levels between WB and qPCR data?
Consider these factors:
In the ariel model, MYH4 L342Q aggregates reduce soluble protein levels, leading to underestimation by WB despite normal mRNA levels .
What strategies enhance MYH4 detection in multiplex cytokine assays?
Biotin’s high affinity for streptavidin necessitates:
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Sequential Detection:
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Signal Amplification:
How to validate MYH4 antibody specificity in human myofibrillar myopathy samples?
Leverage pathological hallmarks:
In ariel homozygotes, MYH4 L342Q forms aggregates detectable only with antibodies recognizing linear epitopes in the rod domain .
Can MYH4 antibodies distinguish fetal vs. adult isoforms in developing muscle?
Temporal specificity requires:
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Developmental Staging:
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Epitope Mapping:
Data from conditional knockout models show that MYH4 antibodies with 1:400 dilution in IHC achieve 95% specificity for adult fast-twitch fibers .
How to standardize MYH4 quantification across muscle fiber types?
Implement normalization protocols:
| Parameter | Protocol Details | Rationale |
|---|---|---|
| Reference Protein | α-actinin (Z-disc marker) | Controls for sarcomere density |
| Signal Normalization | Plot MYH4 intensity per μm² of α-actinin area | Corrects for fiber atrophy |
In diseased muscles, adjust for background using MYH4<sup>-/-</sup> tissue sections incubated with the same antibody lot .
What controls are essential for MYH4 antibody reuse in archived tissues?
Archival stability tests should include:
| Control | Acceptance Criteria | Failure Mode |
|---|---|---|
| Antigen Retrieval | Citrate buffer (pH 6.0, 95°C, 20 min) restores 90% signal | Epitope masking by formalin |
| Lot Consistency | ≤15% CV in staining intensity across 3 lots | Batch-dependent variability |
Boster Bio’s validation data show 12-month stability at -20°C with ≤10% activity loss .
How to troubleshoot MYH4 antibody cross-reactivity in cardiac tissue?
Cardiac-specific challenges arise due to MYH6/7 expression:
| Step | Action | Outcome |
|---|---|---|
| Pre-Absorption | Incubate with 10 μg/mL MYH6 | Eliminates 80% off-target binding |
| Tissue Pre-Treatment | 0.1% Triton X-100 for 30 min | Unmasks MYH4-specific epitopes |
PMC studies confirm that MYH4 antibodies with Biotin conjugation exhibit 5% cross-reactivity to MYH6 in ventricular sections unless pre-absorbed .
