Phospho-MAP3K5 (Ser966) Antibody

What is Phospho-MAP3K5 (Ser966) Antibody and what is its target?

Phospho-MAP3K5 (Ser966) Antibody, also known as Anti-Phospho-ASK1 (S966) antibody, is a polyclonal antibody specifically designed to detect ASK1 (Apoptosis Signal-regulating Kinase 1) protein when phosphorylated at serine residue 966. MAP3K5/ASK1 functions as a component of protein kinase signal transduction cascades, phosphorylating and activating MAP2K4 and MAP2K6, which in turn activate the JNK and p38 MAP kinases respectively . This specific antibody recognizes the phosphorylated form at Ser966, which is an important regulatory site on the protein. The antibody is typically raised in rabbits using a synthesized peptide derived from human ASK1 sequence surrounding the phosphorylation site of Ser966 .

What applications is this antibody validated for?

The Phospho-MAP3K5 (Ser966) antibody has been validated for multiple experimental applications:

The antibody has been specifically verified in various cell lines, including 293 cells treated with TNF (20ng/ml) and calyculinA (50nM) for 15 minutes . When using this antibody for new applications or sample types, it is recommended to perform titration experiments to determine optimal working concentrations for your specific experimental conditions .

What species reactivity has been confirmed?

The Phospho-MAP3K5 (Ser966) antibody has been confirmed to react with human, mouse, and rat MAP3K5 proteins . Cross-reactivity validation has been performed, and no significant cross-reactivity with other proteins has been reported . When working with species other than those listed, validation experiments should be conducted to confirm reactivity before proceeding with full-scale experiments.

How should this antibody be stored and handled?

For optimal performance and longevity, the Phospho-MAP3K5 (Ser966) antibody should be stored at -20°C for long-term preservation (valid for approximately 12 months) . For frequent use over shorter periods (up to one month), the antibody can be stored at 4°C . The antibody is typically supplied in a buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide as preservatives .

To maintain antibody integrity, it is critical to avoid repeated freeze-thaw cycles, which can deteriorate antibody performance . When receiving the antibody (shipped with ice packs), it should be immediately stored at the recommended temperature . For routine use, preparing working aliquots upon first thawing is strongly recommended to minimize freeze-thaw cycles and preserve antibody functionality.

What controls should be used when working with this antibody?

Several important controls should be implemented when working with Phospho-MAP3K5 (Ser966) antibody:

• Positive controls: Lysates from 293 cells treated with TNF (20ng/ml) plus calyculinA (50nM) for 15 minutes have been validated as positive controls .

• Phosphatase treatment control: λ phosphatase-treated NIH/3T3 cells can serve as control samples to demonstrate phospho-specificity .

• Blocking peptide control: Experiments using the phospho-peptide that corresponds to the immunogen can effectively demonstrate antibody specificity. This approach has been validated in both western blot and immunohistochemistry applications .

• Untreated samples: Comparing treated samples with untreated controls helps verify induced phosphorylation.

Including these controls in experimental designs ensures confidence in antibody specificity and experimental validity.

How can Western blot protocols be optimized for this antibody?

Optimizing Western blot protocols for Phospho-MAP3K5 (Ser966) antibody requires attention to several key factors:

• Sample preparation: To preserve phosphorylation status, cell/tissue lysates should be prepared with buffers containing phosphatase inhibitors. The expected molecular weight of MAP3K5/ASK1 is approximately 155 kDa .

• Dilution optimization: Start with the recommended range of 1:500-1:2000 for Western blot applications . A titration experiment can help determine the optimal concentration for your specific sample type.

• Blocking optimization: Standard blocking with 5% BSA in TBST is generally effective, but optimization may be required depending on background issues.

• Incubation conditions: Primary antibody incubation is typically performed overnight at 4°C, but this can be optimized based on signal strength and background.

• Detection system selection: Choose a detection system appropriate for your experimental needs (chemiluminescence, fluorescence, etc.) and expected signal strength.

• Validation with phospho-blocking: Running parallel blots with and without blocking peptide competition can confirm signal specificity, as demonstrated in validation studies where the phospho-peptide successfully blocked antibody binding .

What factors influence the detection of ASK1 phosphorylation at Ser966?

Several factors can influence the detection of ASK1 phosphorylation at Ser966:

• Cell stimulation conditions: Treatments that induce the phosphorylation pathway are essential. TNF-α (20ng/ml) combined with calyculinA (50nM) treatment for 15 minutes has been validated to induce detectable phosphorylation .

• Phosphatase activity: Endogenous phosphatases can rapidly dephosphorylate proteins during sample preparation. Using phosphatase inhibitors (sodium fluoride, sodium orthovanadate, etc.) in lysis buffers is crucial.

• Sample handling: Phosphorylation status can be affected by temperature, time between sample collection and processing, and freeze-thaw cycles.

• Protein extraction methods: Different lysis buffers may vary in their ability to preserve phosphorylation states.

• Cell/tissue type: The baseline phosphorylation level of ASK1 at Ser966 can vary significantly between different cell types and tissues, with notable expression in heart and pancreas tissues .

How does phosphorylation at Ser966 affect ASK1 function in signaling pathways?

Phosphorylation at Ser966 plays a critical regulatory role in ASK1 function and associated signaling pathways. ASK1 (MAP3K5) is a key component in stress-activated signaling that can lead to apoptosis when overexpressed . The Ser966 phosphorylation site is particularly important because:

• This phosphorylation event is often associated with the regulation of ASK1 kinase activity, influencing downstream activation of MAP2K4 and MAP2K6, which subsequently activate JNK and p38 MAPK pathways .

• Phosphorylation status at Ser966 can be modified in response to various cellular stresses, including TNF stimulation, as demonstrated in validation studies using TNF treatment .

• The IRE1-TRAF2-ASK1 complex formation, which is important in stress response signaling, may be influenced by phosphorylation at this site .

Understanding the dynamics of this phosphorylation event can provide insights into how cells regulate stress response pathways and apoptotic signaling under different conditions.

How can this antibody be used for investigating MAP3K5/ASK1 in disease models?

The Phospho-MAP3K5 (Ser966) antibody can be invaluable for investigating the role of ASK1 phosphorylation in various disease models:

• Cancer research: Since ASK1 is involved in apoptotic signaling, its phosphorylation status may be altered in cancer cells. This antibody has been used in breast carcinoma tissue samples, demonstrating its utility in cancer research .

• Cardiovascular research: ASK1 is abundantly expressed in heart tissue , making this antibody useful for investigating phosphorylation changes in cardiovascular disease models, where stress signaling pathways are often dysregulated.

• Neurodegenerative disease models: Stress-activated protein kinase pathways involving ASK1 have been implicated in various neurodegenerative conditions.

• Immunological studies: Given ASK1's response to TNF signaling , this antibody can help dissect immune response mechanisms where ASK1 phosphorylation may be dynamically regulated.

For disease model investigations, combining phospho-specific detection with total ASK1 measurement allows calculation of the phosphorylation ratio, providing insight into the activation status of this pathway in pathological conditions.

What methodological approaches can quantify relative phosphorylation levels?

For quantitative assessment of Ser966 phosphorylation relative to total ASK1 protein levels, several methodological approaches can be employed:

• Dual immunoblotting: Run duplicate samples on separate blots or strip and reprobe with antibodies against phospho-Ser966 ASK1 and total ASK1, then calculate the phospho/total ratio after densitometry.

• Multiplex fluorescent Western blotting: Use differentially labeled secondary antibodies to simultaneously detect phospho-Ser966 ASK1 and total ASK1 on the same blot, enabling direct ratio calculations.

• Quantitative ELISA: Develop a sandwich ELISA approach using the Phospho-MAP3K5 (Ser966) antibody (recommended dilution 1:20000) for capture or detection in combination with total ASK1 measurement.

• Phospho-flow cytometry: For cell-based studies, optimize the antibody for intracellular phospho-flow cytometry to measure phosphorylation levels at the single-cell level.

• Image-based quantification: For IHC applications (using dilutions 1:100-1:300) , digital image analysis can be employed to quantify staining intensity as a measure of phosphorylation levels.

When performing quantitative analyses, calibration standards and consistent experimental conditions are essential for reliable interpretation of phosphorylation dynamics.

What are common issues when working with phospho-specific antibodies?

Several challenges can arise when working with Phospho-MAP3K5 (Ser966) antibody:

• Phosphorylation instability: Phospho-epitopes can be rapidly lost due to phosphatase activity. Immediate sample processing with phosphatase inhibitors is critical.

• Non-specific binding: At incorrect dilutions, phospho-antibodies may show cross-reactivity. Performing careful titration experiments and including appropriate blocking peptide controls can help identify the optimal working concentration .

• Variable phosphorylation levels: Baseline phosphorylation at Ser966 may vary between samples. Using positive controls like TNF+calyculinA treated cells can help establish detection thresholds .

• Epitope masking: In some contexts, protein interactions may mask the phospho-epitope. Testing different sample preparation methods may be necessary.

• Lot-to-lot variability: For polyclonal antibodies like Phospho-MAP3K5 (Ser966) , there may be performance differences between production lots. Validating each new lot against previous results is advisable.

How can researchers verify antibody specificity for phospho-Ser966?

Verifying the specificity of Phospho-MAP3K5 (Ser966) antibody is crucial for experimental rigor:

• Phosphatase treatment: Treating duplicate samples with lambda phosphatase and comparing the signal can confirm phospho-specificity, as demonstrated with NIH/3T3 cells .

• Peptide competition: Using the synthetic phospho-peptide immunogen to block antibody binding should eliminate specific signals, as shown in validation studies with both Western blot and IHC applications .

• Stimulation/inhibition experiments: Compare samples from conditions known to increase (TNF+calyculinA) or decrease Ser966 phosphorylation.

• siRNA or CRISPR knockout controls: Reducing target protein expression should proportionally reduce phospho-specific signal.

• Cross-validation: When possible, verify results using alternative techniques or different antibodies targeting the same phospho-epitope.

Implementing these specificity controls enhances confidence in experimental results and helps distinguish between specific signals and artifacts.