Phospho-FOS (Ser362) Antibody

What is c-Fos and its significance in cellular signaling?

c-Fos is a nuclear phosphoprotein that functions as a critical component of the transcription factor complex AP-1. It belongs to the Fos gene family, which consists of four members: FOS, FOSB, FOSL1, and FOSL2. These proteins contain leucine zipper domains that enable them to dimerize with proteins of the JUN family to form the AP-1 complex . This heterodimeric complex plays a fundamental role in regulating gene expression related to cell proliferation, differentiation, and transformation. The FOS and JUN/AP-1 basic regions interact with symmetrical DNA half-sites to modulate transcription of target genes .

c-Fos has been extensively characterized as a crucial regulator in multiple biological processes, including signal transduction and cellular growth. In growing cells, it activates phospholipid synthesis, potentially through activation of CDS1 and PI4K2A . Additionally, c-Fos has been implicated in skeletal development and maintenance, as it regulates the development of cells destined to form and maintain bone tissue . In certain contexts, expression of the FOS gene has also been associated with apoptotic cell death .

How does phosphorylation at Ser362 affect c-Fos function?

Phosphorylation at Ser362 represents one of several critical post-translational modifications that regulate c-Fos activity and function. While the search results don't provide specific details about the unique effects of Ser362 phosphorylation, we can infer its importance from the existence of specialized antibodies designed to detect this specific modification .

Phosphorylation generally serves as a molecular switch that can alter protein conformation, localization, stability, and interaction capabilities. In the case of c-Fos, phosphorylation at various sites, including Ser362, likely modulates its transcriptional activity, protein-protein interactions, or subcellular localization . The search results indicate that c-Fos phosphorylation can be induced by various stimuli including growth factors and insulin, suggesting that Ser362 phosphorylation may be part of specific signaling cascades responding to these extracellular cues .

Proper storage and handling are crucial for maintaining antibody functionality and specificity. Based on the search results, the consensus for storage of Phospho-FOS (Ser362) Antibody is:

Storage temperature: -20°C is uniformly recommended across all suppliers . For long-term storage, some sources suggest -80°C as an alternative .

Buffer conditions: The antibody is typically supplied in stabilizing buffers containing:

• PBS or TBS with pH ~7.4

• 50% Glycerol in most formulations

• Additional stabilizers such as 0.02% sodium azide

• Some formulations include BSA (0.5-1%)

Handling recommendations:

• Avoid repeated freeze-thaw cycles to prevent protein denaturation and loss of activity

• Aliquot the antibody upon first thaw if multiple uses are anticipated

• Allow the antibody to equilibrate to room temperature before opening the vial

Following these storage guidelines will help ensure optimal antibody performance and extend its useful lifespan in experimental applications .

What validation methods should be employed to confirm the specificity of Phospho-FOS (Ser362) Antibody?

Validation of phospho-specific antibodies requires rigorous controls to ensure specific detection of the phosphorylated form without cross-reactivity to the non-phosphorylated protein. Based on the search results and best practices in antibody validation, researchers should consider the following comprehensive approach:

Phosphatase treatment: Treat one sample with lambda phosphatase to remove phosphate groups, which should eliminate signal from a truly phospho-specific antibody. Compare this with an untreated sample to demonstrate phospho-specificity.

Peptide competition assay: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides corresponding to the region around Ser362. Only the phospho-peptide should block antibody binding in a specific manner .

Knockout/knockdown controls: Use c-Fos knockout or knockdown samples alongside wild-type controls to confirm signal specificity.

Phospho-mimetic and phospho-dead mutants: Express c-Fos constructs with S362A (cannot be phosphorylated) and S362D/E (mimics phosphorylation) mutations to further validate specificity.

Multiple technique comparison: Confirm results across different applications (e.g., WB, IHC, IF) to ensure consistent specificity .

What are the optimal protocols for using Phospho-FOS (Ser362) Antibody in Western blot applications?

Western blot optimization for phospho-specific antibodies requires special consideration to preserve phosphorylation status and maximize signal-to-noise ratio:

Sample preparation:

• Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

• Maintain samples cold throughout processing to prevent dephosphorylation

• Use SDS-PAGE sample buffer with fresh reducing agents

Gel separation and transfer:

• Load adequate protein (typically 20-50 μg of total protein)

• Consider using Phos-tag™ acrylamide gels for enhanced separation of phosphorylated proteins

• Use wet transfer methods with methanol-containing transfer buffer for optimal transfer of phosphoproteins

Blocking and antibody incubation:

• Block with 3-5% BSA in TBST rather than milk (milk contains casein kinases and phosphatases)

• Use recommended dilutions for primary antibody: 1:500-1:2000

• Incubate primary antibody overnight at 4°C for optimal binding

• Use phospho-specific washing protocols (e.g., high salt TBST) to reduce background

Detection:

• Use high-sensitivity ECL reagents appropriate for phospho-protein detection

• Consider signal amplification systems for low-abundance phospho-proteins

Positive controls:

• Include lysates from cells treated with stimuli known to induce c-Fos Ser362 phosphorylation

• Commercial positive control lysates may be available from antibody suppliers

Following these optimized protocols should yield specific detection of c-Fos phosphorylated at Ser362 while minimizing background and non-specific signals.

What upstream kinases are responsible for c-Fos phosphorylation at Ser362?

While the search results don't specifically identify the kinases responsible for Ser362 phosphorylation, they do provide some context about c-Fos phosphorylation in general. The search results mention that c-Fos can be phosphorylated in response to various stimuli, including growth factors and insulin .

For comparison, the search results note that Threonine 232 phosphorylation of c-Fos is mediated by ERK MAPK, which regulates localization of c-Fos to the nucleus and is important for c-Fos induced transcriptional activity . By analogy, Ser362 phosphorylation likely involves specific kinase pathways that respond to cellular stimuli.

Based on the broader scientific literature (though not explicitly stated in the search results), potential candidate kinases for Ser362 phosphorylation may include:

• Members of the MAPK family (ERK, p38, JNK)

• RSK (Ribosomal S6 Kinase)

• MSK (Mitogen and Stress-activated Kinase)

To definitively identify the responsible kinase(s), researchers could employ:

• In vitro kinase assays with purified kinases and c-Fos substrate

• Kinase inhibitor studies examining effects on Ser362 phosphorylation

• Kinase knockdown/knockout approaches

• Phosphorylation site prediction algorithms to generate hypotheses

Understanding the upstream kinase(s) responsible for Ser362 phosphorylation would provide valuable insight into the signaling pathways regulating c-Fos function in specific cellular contexts.

How can researchers troubleshoot non-specific binding when using Phospho-FOS (Ser362) Antibody?

Non-specific binding is a common challenge when working with phospho-specific antibodies. Based on the search results and best practices, here are methodological approaches to troubleshoot such issues:

Optimization of antibody concentration:

• Titrate the antibody across a wider range than recommended (e.g., 1:200-1:5000)

• Use the highest dilution that still produces specific signal

• The search results suggest dilution ranges of 1:500-1:2000 for Western blot applications

Optimization of blocking:

• Test alternative blocking agents (BSA, commercial blockers, casein)

• Increase blocking time or concentration

• Add non-specific IgG from the host species of the secondary antibody

Modification of washing protocol:

• Increase duration and number of wash steps

• Add detergents (0.1-0.3% Triton X-100) to wash buffers

• Use high salt washes (up to 500 mM NaCl) to disrupt low-affinity interactions

Additional specificity controls:

• Use peptide competition with both phosphorylated and non-phosphorylated peptides

• Include phosphatase-treated samples as negative controls

• Use c-Fos knockout/knockdown samples to identify non-specific bands

Technical modifications:

• For IHC/IF: Optimize antigen retrieval methods and fixation protocols

• For WB: Test alternative membrane types (PVDF vs. nitrocellulose)

• Consider using monovalent Fab fragments to block endogenous immunoglobulins in tissue samples

When multiple bands appear in Western blot, determine if they represent:

• Different phosphorylation states of c-Fos

• Proteolytic fragments of c-Fos

• True non-specific binding to unrelated proteins

The high purity (>95% by SDS-PAGE) reported for some commercial versions of this antibody suggests that non-specific binding issues might be minimized with proper optimization .

What are the different epitope designs for Phospho-FOS (Ser362) Antibody and their implications?

The search results reveal variations in the epitope design for Phospho-FOS (Ser362) antibodies across different manufacturers, which can impact specificity, sensitivity, and application performance:

Epitope length and location:

• Most manufacturers use peptides derived from the region surrounding Ser362

• The common amino acid range is 331-380 of human c-Fos

• One source specifically mentions the sequence "K-G-S(p)-S-S" around the phosphorylation site

Immunogen conjugation:

• KLH (Keyhole Limpet Hemocyanin) conjugation is commonly used

• Synthetic phosphopeptide derivation is the standard approach

These epitope design differences can impact:

Specificity: Longer epitopes (e.g., the 331-380 range) may increase specificity by providing more context around the phosphorylation site, but could potentially increase the risk of cross-reactivity with related proteins containing similar sequences.

Sensitivity: The immediate sequence context (K-G-S(p)-S-S) may provide optimal exposure of the phospho-epitope, potentially increasing sensitivity.

Cross-reactivity: The high conservation of this region across species explains the broad species reactivity reported .

Researchers should consider these epitope design factors when selecting an antibody for specific applications. For instance, antibodies with longer epitopes might be preferable for applications requiring high specificity, while those with optimized immediate context around the phosphorylation site might be better for detecting low-abundance phospho-proteins.

How can Phospho-FOS (Ser362) Antibody be optimized for immunohistochemistry applications?

Optimizing immunohistochemistry protocols for phospho-specific antibodies requires special considerations to preserve phosphorylation epitopes and enhance specific detection:

Tissue fixation and processing:

• Use phosphatase inhibitor-containing fixatives

• Minimize fixation time (4-24 hours in 10% neutral buffered formalin is often optimal)

• Process tissues rapidly to avoid phospho-epitope degradation

Antigen retrieval optimization:

• Compare heat-induced epitope retrieval methods:

  • Citrate buffer (pH 6.0)

  • EDTA buffer (pH 9.0)

  • Tris-EDTA (pH 9.0)

• Include phosphatase inhibitors in retrieval solutions

• Optimize retrieval time (typically 10-30 minutes)

Blocking and antibody incubation:

• Use recommended dilutions for IHC-P (1:100-1:400) and IHC-F (1:100-1:500)

• Consider longer primary antibody incubation (overnight at 4°C)

• Use antibody diluent containing phosphatase inhibitors

Signal amplification:

• Consider tyramide signal amplification for low-abundance phospho-proteins

• Polymer detection systems often provide better sensitivity than ABC methods

Controls:

• Adjacent sections treated with/without lambda phosphatase

• Tissue known to express/not express phosphorylated c-Fos

• Peptide competition controls with phospho and non-phospho peptides

The search results indicate that Phospho-FOS (Ser362) Antibody has been validated for both paraffin-embedded and frozen tissue sections , suggesting flexibility in sample preparation approaches. By systematically optimizing these parameters, researchers can achieve specific and sensitive detection of phosphorylated c-Fos in tissue samples.

What considerations are important when designing experiments to study c-Fos phosphorylation dynamics?

Studying the dynamics of c-Fos phosphorylation requires careful experimental design to capture temporal and spatial regulation. Based on the search results and established principles in signaling research:

Temporal considerations:

• Establish appropriate time courses to capture rapid phosphorylation/dephosphorylation events

• Include early time points (minutes) to capture immediate phosphorylation events

• Include later time points (hours) to observe potential feedback regulation

• The search results mention that c-Fos can be phosphorylated in response to various stimuli including growth factors and insulin

Stimulation protocols:

• Standardize stimulation conditions (concentration, duration, temperature)

• Include appropriate vehicle controls

• Consider physiologically relevant stimulation intensities

• Use multiple stimuli to compare pathway-specific phosphorylation patterns

Sample preparation:

• Rapidly lyse cells/tissues in buffers containing phosphatase and protease inhibitors

• Maintain cold temperatures throughout processing

• Consider subcellular fractionation to track phosphorylation-dependent localization changes

• The nuclear localization of c-Fos may necessitate specific nuclear extraction protocols

Quantification methods:

• Use quantitative approaches (Western blot densitometry, ELISA, phospho-flow cytometry)

• Normalize phospho-signals to total c-Fos protein levels

• Consider multi-plexed approaches to simultaneously monitor multiple phosphorylation sites

• The molecular weight of c-Fos is reported as approximately 40-62 kDa , which helps in correctly identifying the target band

When working with heterogeneous tissue or cell populations, consider single-cell approaches like phospho-flow cytometry or immunofluorescence to resolve cell-type specific phosphorylation events. The search results indicate that Phospho-FOS (Ser362) Antibody supports multiple detection methodologies, enabling flexible experimental design .

How does phosphorylation at Ser362 interact with other post-translational modifications of c-Fos?

While the search results don't provide specific information about interactions between Ser362 phosphorylation and other post-translational modifications (PTMs), this represents an important research question. Based on general principles of PTM crosstalk and the available information about c-Fos:

Potential PTM interactions:

• Hierarchical phosphorylation: Phosphorylation at one site can prime for or inhibit phosphorylation at another site

• The search results mention that c-Fos can be phosphorylated at multiple sites (at least seven different sites)

• Phosphorylation-dependent ubiquitination: Some phosphorylation events can trigger ubiquitination and subsequent degradation

• Phosphorylation-dependent protein interactions: Phosphorylation can create or disrupt binding sites for interacting proteins

Methodological approaches to study PTM crosstalk:

• Mass spectrometry-based proteomics to identify co-occurring modifications

• Site-directed mutagenesis of individual or multiple phosphorylation sites

• Antibodies specific to different phosphorylation sites used in combination

• Kinase and phosphatase inhibitors to manipulate specific phosphorylation events

The search results mention that c-Fos forms a complex with JUN/AP-1 , and this interaction could potentially be regulated by phosphorylation at Ser362. Additionally, the activity of c-Fos in TGF-beta signaling involves formation of a multimeric SMAD3/SMAD4/JUN/FOS complex , which might also be influenced by phosphorylation status.

Future research directions could include investigating how Ser362 phosphorylation affects:

• Protein stability and half-life

• Nuclear localization

• Interaction with JUN family proteins

• DNA binding affinity

• Recruitment of transcriptional co-activators or co-repressors

Understanding these interactions would provide valuable insight into how c-Fos activity is fine-tuned through combinatorial post-translational modifications.

Future Research Directions and Emerging Applications

The Phospho-FOS (Ser362) Antibody represents a valuable tool for investigating the complex regulatory mechanisms governing c-Fos function in diverse cellular contexts. Based on the search results and current research trends, several promising future directions emerge:

Integration with high-throughput technologies:

• Single-cell phospho-proteomics to resolve cell-type specific c-Fos phosphorylation patterns

• ChIP-seq following stimulation to map phosphorylation-dependent genomic binding sites

• Proximity labeling approaches to identify phosphorylation-dependent protein interactions

Therapeutic relevance exploration:

• Investigating c-Fos Ser362 phosphorylation in pathological contexts

• The oncogenic potential of c-Fos suggests potential relevance in cancer research

• The role of c-Fos in skeletal development indicates possible applications in bone-related disorders

Technical innovations:

• Development of biosensors to monitor c-Fos phosphorylation in live cells

• Nanobody-based detection reagents for improved spatial resolution

• Targeted degradation approaches to selectively remove phosphorylated c-Fos species

The continued refinement of antibody specificity, combined with advances in imaging and proteomics technologies, will enable increasingly sophisticated investigations of c-Fos phosphorylation dynamics. As our understanding of the functional consequences of Ser362 phosphorylation deepens, new opportunities for therapeutic intervention in c-Fos-related pathologies may emerge.