Phospho-DES (S60) Antibody
Description
General Definition and Purpose of Phospho-Site-Specific Antibodies
Phosphorylation state-specific antibodies (PSSAs) are tools designed to detect proteins phosphorylated at specific serine, threonine, or tyrosine residues. These antibodies enable researchers to study dynamic phosphorylation events critical for cellular signaling, disease mechanisms, and therapeutic targeting .
Key Features of Phospho-S60 Antibodies (Analogous Systems)
While DES (Desmin) phosphorylation at Ser60 is not described in the provided sources, insights can be drawn from studies on other S60-phosphorylated proteins:
Example 1: β-Catenin p-S60 Antibody
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Target: β-catenin phosphorylated at Ser60.
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Role: Facilitates cytokinesis by recruiting Ect2 to the midbody, activating RhoA, and promoting actomyosin contractility .
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Validation:
Example 2: α-Synuclein p-S60 Antibody
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Target: α-synuclein phosphorylated at Ser60 in Nothobranchius furzeri (turquoise killifish) models.
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Role: Associated with aging and neurodegenerative pathologies.
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Validation:
Development and Validation of Phospho-Specific Antibodies
Potential Applications for a Hypothetical Phospho-DES (S60) Antibody
If developed, a Phospho-DES (S60) antibody could:
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Investigate Desmin’s role in muscle cell contractility or cytoskeletal dynamics.
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Study diseases linked to Desmin phosphorylation, such as myopathies or cardiomyopathies.
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Validate kinase/phosphatase pathways targeting Desmin Ser60.
Challenges and Considerations
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Cross-Reactivity: Antibodies may bind non-specifically to similar phospho-epitopes in other proteins .
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Epitope Stability: Phospho-epitopes are labile; tissue fixation and processing protocols must preserve modifications .
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Validation: Requires orthogonal methods (e.g., mass spectrometry, genetic knockouts) to confirm specificity .
Product Specs
Target Background
- A novel mutation (c.679 C>T /p.R227C) in exon 3 of DES was identified and cosegregated with affected members of a Chinese family exhibiting isolated Dilated cardiomyopathy (DCM) phenotypes (left ventricle and left atrial diameters). PMID: 28171858
- Desmin, Glial Fibrillary Acidic Protein, Vimentin, and Peripherin are type III intermediate filaments that play roles in both health and disease. [review] PMID: 29196434
- Phenotypic expression of a novel desmin gene mutation: hypertrophic cardiomyopathy followed by systemic myopathy. PMID: 29167554
- Targeted sequencing revealed trigenic mutations: c.700G>A/p.E234K in DES, c.2966G>A/p.R989H in MYPN, and c.5918G>C/p.R1973P in CACNA1C in a family with hypertrophic cardiomyopathy, early repolarization, and short QT syndrome. PMID: 28427417
- Research demonstrates that the expression of mutant desmin disrupts the extrasarcomeric desmin cytoskeleton and leads to significant mitochondrial abnormalities in terms of subcellular distribution, number, and shape. PMID: 27393313
- Mutation in the Core Structure of Desmin Intermediate Filaments Affects Myoblast Elasticity PMID: 28793217
- Data indicates that the filament elongation of both desmin and keratin K8/K18 proceeds very similar to that of vimentin. PMID: 27304995
- Cdk1-induced desmin phosphorylation is essential for efficient separation of desmin-IFs and is generally detected in muscular mitotic cells in vivo. PMID: 27565725
- Desmin, Lamin A/C, MMP9, and histone H4 were found to be upregulated in the placental villi of women experiencing early pregnancy loss. PMID: 26947931
- Ile367Phe, Pro419Ser, and Arg415Glu mutations were associated with desminopathy causing cardiomyopathy in four families studied. PMID: 26431784
- An increase in desmin abnormalities was correlated with the progression of diastolic dysfunction. PMID: 25732530
- Studies compared the expression level of mutant versus wild-type desmin in a mouse model and in skeletal muscle specimens from human R350P desminopathies. The findings demonstrate that missense-mutant desmin induces changes in the subcellular localization and turnover of desmin itself and its direct binding partners. PMID: 25394388
- Results suggest that the mutations affect desmin structure, causing aberrant folding and subsequent aggregation, ultimately leading to disruption of myofibrils organization. PMID: 25541946
- Researchers identified disruption of the desmin system in gastrocnemius myofibers as an indicator of myopathy and limited muscle function in patients with peripheral artery disease. PMID: 25575565
- The desmin intermediate filament network plays a significant role in striated muscle development and maintenance by integrating and coordinating essential cellular components for proper mechanochemical signaling, organelle communication, energy production, and trafficking processes necessary for tissue homeostasis. [Review] PMID: 25680090
- Data suggests that loss of desmin-p. A120D filament localization at the intercalated disk indicates its clinical arrhythmogenic potential. PMID: 24200904
- Researchers describe a new mutation located in the coiled 1B domain of desmin associated with predominant cardiac involvement and a high incidence of cardiac sudden death in a large Indian pedigree with 12 affected members. PMID: 24441330
- Proteomic analysis conducted on a transgenic mouse model of severe cardiac hypertrophy and compared to a heart failure dataset identified MYH7, IGFBP7, ANXA2, and DESM as potential biomarker candidates for heart failure. PMID: 23713052
- Autosomal recessive mutations in DES cause LGMD2 phenotype without features of myofibrillar myopathy. PMID: 23687351
- Data suggests that for certain filament-forming desmin mutants, the molecular basis of desminopathy arises from subtle deficiencies in their association with nebulin, a major actin-binding filament protein in striated muscle. PMID: 23615443
- Sequencing of the desmin gene revealed a splice-site mutation (IVS3+1G-->A), which was absent in 300 healthy control subjects. PMID: 22484823
- Phenotypic features in patients with desmin tail domain mutations resemble those observed in patients with mutations localized in the 1B and 2B alpha-helical domains. PMID: 23051780
- The findings of this study indicate that atrioventricular conduction block without cardiac structural abnormalities might be an intrinsic characteristic of disease associated with specific desmin mutations. PMID: 23036309
- In the absence of skeletal muscle involvement suggesting a desminopathy, the likelihood of DES mutations in ARVC is very low. This finding has significant implications for the mutation screening strategy for patients with ARVC. PMID: 23168288
- The results of this study indicated that no cases exhibited missense mutations in Desmin. PMID: 22349865
- Data reveals that calretinin and CK5/6 were positive in 100% and 64% of mesotheliomas, respectively, and 92% and 31% of reactive effusions, respectively. Desmin was negative in all malignant cases and positive in 85% of reactive effusions. PMID: 23075894
- A heterozygous C-to-T mutation in the desmin gene on chromosome 2q35 caused autosomal dominant myofibrillar myopathy with arrhythmogenic right ventricular cardiomyopathy 7 in a Swedish family. PMID: 22395865
- Frequent desmin (32%) and occasional CD34 (6%) expression are observed in cellular fibrous histiocytoma. PMID: 22775584
- Data indicates that the interaction and co-localization of mutant and wild-type desmin demonstrate the coexistence of heterogeneous filaments in living cells. PMID: 22403400
- Analysis of CD105, CD31, alpha-SMA, vimentin, and desmin expression on a series of normal human heart tissues ranging from five to 33 weeks. PMID: 22395512
- This study demonstrated that patients carrying DES mutations presenting with myofibrillar myopathies or without muscle involvement are at a high risk of developing significant cardiac complications. PMID: 22153487
- The expression of mutant desmin leads to increased mechanical stiffness, resulting in excessive mechanical stress in response to strain and consequently increased mechanical vulnerability and damage to muscle cells. PMID: 22386993
- Ankrd1 and desmin may play important roles in airway smooth muscle cell homeostasis. PMID: 22085644
- Forty-nine mutations have been identified in the desmin gene, which alter the desmin filament assembly process through various molecular mechanisms and its interaction with its protein partners. Review. PMID: 21982405
- These studies highlight the importance of desmin in maintaining cardiomyocyte structure and illustrate how disrupting this network can be detrimental to the heart. --{REVIEW} PMID: 21784990
- Study of patients with heart dilation of various origins; conclude A213V desmin substitution represents a rare polymorphism that acts as a predisposing factor resulting in maladaptive heart remodeling in the presence of other pathological factors. PMID: 21842594
- Novel mutations of the desmin gene were linked with cardiomyopathy in patients from five Chinese families with desminopathy. PMID: 20654101
- Six novel mutations and one previously reported mutation in the desmin gene were identified in the patients. PMID: 20696008
- The state of desmin-filament assembly is crucial for synemin anchorage and consequently might involve the mechanical and functional stability of the cytoskeletal network. PMID: 21262226
- Study provides evidence on the functional consequences of a novel mutation, N116S, identified in the desmin 1A segment of the rod domain for the development of arrhythmogenic right ventricular cardiomyopathy. PMID: 20829228
- Case Report: presents a rare case of desmin-related hypertrophic cardiomyopathy. Cardiac magnetic resonance imaging revealed fibrosis in the lateral wall of the left ventricle. PMID: 21083940
- Mutations in MTM1 disrupted the MTM1-desmin complex, resulting in abnormal intermediate filament assembly and architecture in muscle cells. PMID: 21135508
- Desmin mutations affect the localization of desmoplakin and plakophilin-2 at the intercalated disk, suggesting a link between desmosomal cardiomyopathies (mainly affecting the right ventricle) and cardiomyopathies caused by desmin mutations. PMID: 20423733
- Study of aggregation properties of desmin in vitro & aggregation state of desmin in homogenates of transfected cells; detected divergent assembly patterns for three different desmin missense mutations. PMID: 20448486
- The Uruguayan family with severe cardiomyopathy carries an unusual deletion p.E114del within the 1A rod domain of desmin. PMID: 20133133
- The "tail" domain is responsible for attractive filament-filament interactions. PMID: 20171226
- Mutations in the "head" region of desmin proteins, impacting intermediate filament assembly properties and their competition for binding to cellular anchoring structures, may explain part of the molecular mechanism causing myofibrillar myopathy disease. PMID: 19763525
- A localized effect of desmin on the structure of the cardiac intercalated disks might contribute to disease pathogenesis. PMID: 19879535
- Data showed that the elevated expression of desmin was correlated with the severity and differentiation of CRC. PMID: 19460759
- Structural and functional analysis of a new variant causing desmin-related myopathy. PMID: 11668632
Q&A
What is Phospho-DES (S60) Antibody and what does it specifically detect?
Phospho-DES (S60) antibody is a phospho-specific antibody that recognizes desmin protein only when phosphorylated at the serine 60 residue. Desmin is an intermediate filament protein that plays a crucial role in stress transmission and mechano-protection in muscle cells . This antibody allows researchers to differentiate between phosphorylated and non-phosphorylated forms of desmin at this specific site.
The antibody is typically generated using a synthetic phosphopeptide derived from human desmin around the phosphorylation site of S60 . The specificity for the phosphorylated form is achieved through affinity purification techniques that remove antibodies that might bind to non-phosphorylated forms .
What experimental techniques can effectively utilize Phospho-DES (S60) Antibody?
The Phospho-DES (S60) antibody can be employed in multiple experimental techniques:
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Western Blotting: For quantitative analysis of phosphorylation levels in whole cell lysates, supernatants, or pellet fractions .
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Immunohistochemistry (IHC): To visualize the distribution and localization of phosphorylated desmin in tissue sections .
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Immunoprecipitation: To isolate phosphorylated desmin complexes from cell or tissue lysates .
| Application | Typical Dilution | Sample Preparation Considerations |
|---|---|---|
| Western Blot | 1:1000 - 1:2000 | Phosphatase inhibitors critical; subcellular fractionation may be required |
| IHC | 1:100 - 1:300 | Antigen retrieval optimization; phospho-epitope preservation |
| ELISA | 1:5000 - 1:20000 | Standard curves using phospho-peptides |
How should samples be prepared to maintain phosphorylation status for Phospho-DES (S60) detection?
Sample preparation is critical for accurately detecting phosphorylation states:
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Immediate preservation: Tissues should be snap-frozen in liquid nitrogen immediately after collection to prevent phosphatase activity .
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Lysis buffer composition: Use buffers containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) to prevent dephosphorylation during extraction .
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Subcellular fractionation: For desmin specifically, sequential extraction into TBS-soluble and TBS-insoluble/SDS-soluble fractions may be necessary to analyze compartmentalized phosphorylation patterns .
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Temperature control: Process samples at 4°C to minimize enzymatic activity that could alter phosphorylation status.
What is the physiological relevance of Desmin phosphorylation at S60?
Desmin phosphorylation at S60 has significant physiological implications:
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Baseline phosphorylation: Contrary to earlier beliefs that desmin phosphorylation primarily occurred in pathological conditions, research now confirms that S60 phosphorylation exists as a regular physiological mechanism in healthy muscle tissue .
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Subcellular localization: Phosphorylated desmin at S60 is primarily localized in the supernatant (cytosolic) fraction rather than in the pellet (filamentous) fraction, suggesting that this modification may affect protein solubility and assembly properties .
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Response to training: Resistance training decreases baseline phosphorylation at S60, suggesting adaptation mechanisms that may stabilize the intermediate filament system .
How does acute resistance exercise affect Desmin S60 phosphorylation?
Acute resistance exercise produces significant changes in desmin phosphorylation:
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Immediate dephosphorylation: Following acute resistance exercise, there is a significant reduction in phosphorylation at the S60 site .
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Protective mechanism: This dephosphorylation may represent a protective mechanism to stabilize the intermediate filament system during mechanical stress, preventing destabilization during increased mechanical loads .
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Fiber-type specificity: Type I muscle fibers display higher levels of phosphorylated desmin at S60 compared to type II fibers, reflecting fiber-specific regulation mechanisms .
How can I validate the specificity of a Phospho-DES (S60) Antibody?
Antibody validation is crucial for phospho-specific research. The following methods should be employed:
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Peptide competition assays: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides to confirm specificity for the phosphorylated form .
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Phosphatase treatment: Treat half of your sample with phosphatases (e.g., calf intestinal phosphatase - CIP) to remove phosphate groups and confirm loss of signal .
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Site-directed mutagenesis: Use samples expressing S60A (serine to alanine) mutants that cannot be phosphorylated as negative controls .
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Knockout controls: Where possible, use desmin-deficient samples to confirm absence of signal .
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Multiple detection methods: Confirm findings across Western blot, immunohistochemistry, and other techniques to ensure consistent results .
How can dynamic changes in Desmin S60 phosphorylation be monitored in response to exercise or pathological conditions?
To effectively track dynamic phosphorylation changes:
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Time course experiments: Design sampling protocols that capture both immediate (1 hour post-exercise) and delayed (12-24 hours) changes in phosphorylation status .
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Subcellular fractionation combined with Western blotting: This approach can reveal shifts between soluble and insoluble pools of phosphorylated desmin .
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Quantitative comparisons across conditions: Use standardized loading controls and normalization methods to accurately quantify relative phosphorylation levels between conditions.
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Phosphoproteomic approaches: For comprehensive analysis, consider mass spectrometry-based phosphoproteomics to identify multiple phosphorylation sites simultaneously .
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Using phosphomimetic mutants: Experimentally, S60D or S60E mutations can be used to mimic constitutive phosphorylation for functional studies .
What is the relationship between S60 phosphorylation and other post-translational modifications of Desmin?
Understanding the interplay between multiple modifications requires sophisticated approaches:
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Multi-phosphosite analysis: Desmin can be phosphorylated at multiple sites including S31, S60, T17, and T76/77, each with distinct subcellular localizations and functional implications .
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Cross-talk mechanisms: Evidence suggests that phosphorylation at one site may influence modification at others, creating complex regulatory networks .
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Other PTM interactions: O-GlcNAcylation may interact with phosphorylation, particularly for sites like S60 that are exclusively found in the cytosolic fraction .
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Methodological approach: Use a panel of phospho-specific antibodies targeting different sites to map the phosphorylation landscape under various conditions .
| Phosphorylation Site | Subcellular Localization | Response to Acute Exercise | Response to Training |
|---|---|---|---|
| S31 | Mainly pellet fraction | Dephosphorylation | Increased baseline phosphorylation |
| S60 | Exclusive to supernatant | Dephosphorylation | Decreased baseline phosphorylation |
| T17 | Exclusive to supernatant | No change | Decreased baseline phosphorylation |
| T76/77 | Both pellet and supernatant | No change | No change |
How do phosphorylation patterns of Desmin at S60 differ between normal physiology and pathological conditions?
This question requires sophisticated comparative analysis:
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Normal physiological regulation: In healthy muscle, S60 phosphorylation is part of normal cytoskeletal regulation and responds dynamically to exercise stimuli .
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Pathological implications: Increased intermediate filament phosphorylation, including at sites like S60, has been associated with IF-system destabilization in conditions such as ischemic heart failure and catabolic states .
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Methodological considerations for comparative studies:
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Use matched controls and standardized extraction protocols
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Account for fiber-type differences when comparing pathological and healthy samples
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Consider time course of disease progression and adaptive responses
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Mechanistic insights: The difference between physiological and pathological phosphorylation may lie not in the mere presence of phosphorylation but in its temporal dynamics and coordination with other regulatory mechanisms .
What methodological challenges exist when analyzing phosphorylation dynamics across different cellular compartments?
Advanced compartmental analysis presents several challenges:
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Epitope masking: The monoclonal anti-total desmin antibody may not recognize all forms of desmin due to epitope masking by post-translational modifications .
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Fractionation protocols: Different extraction buffers and sequential extraction methods can yield varying results for phosphorylated protein recovery .
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Solubility changes: Phosphorylation itself can alter protein solubility, potentially causing shifts between fractions independent of localization changes .
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Technical solution: Use multiple antibodies raised against different epitopes to ensure comprehensive detection, and validate findings with immunofluorescence microscopy to confirm subcellular localization .
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Data integration: Combine biochemical fractionation data with imaging studies for a more complete understanding of spatial dynamics.
How can Phospho-DES (S60) antibodies be integrated into multi-parameter signaling studies?
For comprehensive signaling analysis:
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Phospho-antibody arrays: Incorporate Phospho-DES (S60) into antibody arrays containing multiple phospho-specific antibodies to analyze coordinated signaling networks .
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Multiplex immunofluorescence: Use spectrally distinct fluorophores to simultaneously detect multiple phosphorylation sites on desmin and related proteins in tissue sections .
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Integration with kinase/phosphatase assays: Combine phospho-detection with activity assays for relevant kinases and phosphatases to understand regulatory mechanisms .
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Systems biology approach: Correlate S60 phosphorylation data with transcriptomic and proteomic datasets to identify regulatory networks .
What are the considerations for developing and validating new phospho-specific antibodies for novel sites on desmin or related intermediate filament proteins?
Researchers developing new phospho-specific antibodies should consider:
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Epitope selection: Choose sequences with high antigenicity and minimal similarity to other phosphorylation sites .
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Immunization strategies: Use carrier-conjugated phosphopeptides with complete adjuvants followed by boosting with incomplete adjuvants .
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Purification approach: Employ sequential affinity purification using both phospho-peptide and non-phospho-peptide columns to isolate truly phospho-specific antibodies .
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Validation requirements:
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Cross-species reactivity: Design considering sequence conservation if cross-species application is desired .
How can computational approaches enhance the interpretation of Phospho-DES (S60) data in the context of cytoskeletal dynamics?
Advanced computational methods can provide deeper insights:
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Structural modeling: Predict how S60 phosphorylation affects desmin filament assembly and interactions with other proteins based on structural data.
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Kinetic modeling: Develop mathematical models of phosphorylation/dephosphorylation cycles to predict temporal dynamics following exercise stimuli.
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Network analysis: Integrate phosphorylation data with protein-protein interaction networks to identify regulatory hubs and feedback mechanisms.
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Machine learning applications: Apply pattern recognition algorithms to identify subtle correlations between phosphorylation patterns and functional outcomes across different experimental conditions.
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Image analysis algorithms: Develop specialized tools for quantifying spatial distribution of phosphorylated desmin from immunofluorescence data, particularly relevant for studying fiber-type specific effects .
