Phospho-CAT (Y386) Antibody

What is Phospho-CAT (Y386) Antibody and what does it detect?

Phospho-CAT (Y386) Antibody is a polyclonal antibody that specifically recognizes the catalase enzyme when it is phosphorylated at tyrosine residue 386. Catalase (CAT) is a critical antioxidant enzyme with a molecular weight of approximately 60 kDa that catalyzes the decomposition of hydrogen peroxide to water and oxygen. The antibody is designed to detect endogenous levels of the catalase protein only when the specific tyrosine residue is phosphorylated, allowing researchers to study post-translational modifications that may affect enzyme function .

What are the validated applications for Phospho-CAT (Y386) Antibody?

Phospho-CAT (Y386) Antibody has been validated for multiple experimental applications, including:

• Western Blotting (WB) at dilutions of 1:500-1:2000

• Immunohistochemistry (IHC) at dilutions of 1:100-1:300

• Enzyme-Linked Immunosorbent Assay (ELISA) at dilutions of 1:20000

These applications allow researchers to detect the phosphorylated form of catalase in cell lysates, tissue sections, and purified protein preparations, providing versatility in experimental design .

What species reactivity has been confirmed for this antibody?

The Phospho-CAT (Y386) Antibody demonstrates confirmed cross-reactivity with human, mouse, and rat samples. This multi-species reactivity makes it valuable for comparative studies across different model organisms, enabling researchers to investigate evolutionary conservation of catalase phosphorylation and its biological significance .

How should phosphorylation-positive control samples be prepared for validating Phospho-CAT (Y386) Antibody?

For preparing phosphorylation-positive control samples, researchers can employ a simple in vitro phosphorylation system without the need for live cells or protein purification:

• Harvest cell or tissue lysates and place in an ice bath for 20 minutes (homogenize tissues before incubation)

• Treat lysates with ultrasound and centrifuge at 12,000g, 4°C for 15 minutes

• Add ATP to the supernatant at a final concentration of 5 mM

• Incubate the mixture in a water bath at 30°C for 30 minutes

• Use the resulting supernatant for Western blotting or ELISA

This method allows for the generation of phosphorylation-positive controls that can help validate the specificity of the Phospho-CAT (Y386) Antibody and establish appropriate experimental conditions .

What protocol modifications are necessary for immunofluorescence experiments with Phospho-CAT (Y386) Antibody?

For immunofluorescence experiments, the following protocol is recommended:

• Treat cells with the in vitro phosphorylation buffer in a CO₂ incubator at 37°C for 40 minutes

• Discard the buffer and wash cells three times with 1× TBS

• Fix cells in a 1:1 mixture of methanol and acetone at 4°C for 20 minutes

• Wash three times with PBST (PBS containing Tween)

• Block with 5% BSA in PBST at room temperature for 30 minutes

• Incubate with Phospho-CAT (Y386) Antibody (1:100 dilution) for 4 hours at room temperature

• Wash three times with PBST

• Incubate with fluorophore-conjugated secondary antibody for 45 minutes at 37°C

• Counterstain with DAPI for 5 minutes at 37°C

• Wash with methanol three times and PBS three times

• Analyze using confocal microscopy

This protocol ensures optimal detection of phosphorylated catalase in cellular contexts while minimizing background signal .

How does the phosphorylation status of catalase influence antibody selection for experimental design?

When designing experiments to study catalase, researchers must carefully consider whether total catalase or specifically the phosphorylated form is the target of interest. Phosphorylation at Y386 may regulate catalase activity or protein-protein interactions. If studying phosphorylation-dependent functions, a phospho-specific antibody like Phospho-CAT (Y386) is essential.

For comprehensive studies, researchers often employ both phospho-specific and non-phospho-specific antibodies in parallel experiments to determine:

• Total catalase expression levels (using non-phospho antibodies)

• Proportion of catalase that is phosphorylated (using phospho-specific antibodies)

• Changes in phosphorylation status under different experimental conditions

This dual approach provides critical insights into how post-translational modifications affect catalase function in response to various stimuli or in different disease states .

How can researchers quantitatively analyze Western blot data from Phospho-CAT (Y386) Antibody experiments?

Quantitative analysis of Western blot data from Phospho-CAT (Y386) Antibody experiments should follow these methodological steps:

• Normalize phospho-CAT signal to an appropriate loading control (e.g., β-actin, GAPDH)

• Calculate the ratio of phosphorylated to total catalase by running parallel blots with phospho-specific and total catalase antibodies

• Use densitometry software to quantify band intensity, ensuring analysis is performed within the linear range

• Present data as fold-change relative to control conditions

• Apply appropriate statistical tests (typically t-test for two groups or ANOVA for multiple groups)

For accurate interpretation, researchers should also consider:

• The molecular weight of catalase (~60 kDa) to confirm antibody specificity

• Potential changes in total catalase expression that might influence phosphorylation ratios

• The dynamic range of the detection method to avoid signal saturation .

What factors should be considered when interpreting discrepancies between phospho-specific antibody results and other phosphorylation detection methods?

When encountering discrepancies between results obtained with Phospho-CAT (Y386) Antibody and other phosphorylation detection methods (e.g., mass spectrometry), researchers should consider several potential factors:

• Antibody specificity: Cross-reactivity with similar phosphorylation motifs in other proteins

• Sensitivity thresholds: Different methods have varying detection limits

• Sample preparation differences: Phosphatase activity during sample preparation may reduce detectable phosphorylation

• Stoichiometry of phosphorylation: Low-abundance phosphorylation may be detectable by enrichment methods but not by direct antibody detection

• Spatial resolution: Mass spectrometry provides site-specific information that may be more precise than antibody recognition

To resolve discrepancies, researchers should validate findings through multiple approaches:

• Peptide competition assays to confirm antibody specificity

• Phosphatase treatment of samples as negative controls

• Correlation with functional assays to determine biological relevance

• Use of phosphomimetic (Y386D) and phospho-null (Y386F) mutants .

How can phospho-flow cytometry complement Western blot analysis when using Phospho-CAT (Y386) Antibody?

Phospho-flow cytometry provides a powerful complementary approach to Western blot analysis when studying catalase phosphorylation:

The methodology for phospho-flow cytometry with Phospho-CAT (Y386) Antibody involves:

• Stimulate cells under experimental conditions

• Fix and permeabilize cells to allow antibody access

• Stain with Phospho-CAT (Y386) Antibody and fluorophore-conjugated secondary antibody

• Analyze by flow cytometry

This approach allows researchers to:

• Determine heterogeneity in catalase phosphorylation within cell populations

• Correlate phosphorylation status with other cellular parameters

• Track dynamic changes in phosphorylation in response to stimuli .

How does catalase phosphorylation at Y386 influence its enzymatic activity and what experimental approaches can measure this relationship?

The phosphorylation of catalase at Y386 may significantly impact its enzymatic activity through several potential mechanisms:

• Alteration of protein conformation affecting active site accessibility

• Changes in protein-protein interactions or subcellular localization

• Modification of substrate binding affinity

• Influence on enzyme stability or turnover rate

To experimentally measure this relationship, researchers can employ:

• Enzymatic activity assays: Compare the H₂O₂ decomposition rate between phosphorylated and non-phosphorylated catalase using:

  • Spectrophotometric assays monitoring H₂O₂ consumption at 240 nm

  • Amplex Red-based fluorometric assays for enhanced sensitivity

  • Oxygen electrode measurements to detect O₂ production

• Site-directed mutagenesis: Generate Y386F (phospho-null) and Y386D/E (phosphomimetic) mutants to simulate permanent non-phosphorylated and phosphorylated states, respectively

• In vitro phosphorylation/dephosphorylation: Treat purified catalase with specific kinases and phosphatases while monitoring activity changes

• Structural studies: Use X-ray crystallography or molecular dynamics simulations to visualize how phosphorylation affects protein structure .

What signaling pathways regulate catalase phosphorylation at Y386 and how can they be experimentally manipulated?

Catalase phosphorylation at Y386 is likely regulated by specific tyrosine kinases and phosphatases within signaling pathways that respond to oxidative stress, growth factors, or inflammatory stimuli. While the exact pathways are still being elucidated, researchers can experimentally manipulate and study these regulatory mechanisms through:

• Kinase inhibitor screening: Treat cells with a panel of specific kinase inhibitors (e.g., SRC family, JAK, EGFR inhibitors) and assess changes in catalase Y386 phosphorylation

• siRNA/CRISPR-based knockdown/knockout: Systematically target candidate kinases and phosphatases to identify regulators of catalase phosphorylation

• Phosphatome profiling: Use phosphatase assays and proteomics to identify phosphatases that act on phospho-Y386

• Stimulus-response experiments: Expose cells to oxidative stress inducers (H₂O₂, paraquat), growth factors (EGF, PDGF), or inflammatory cytokines (TNF-α, IL-6) and monitor catalase phosphorylation kinetics

• Pathway reconstruction: Reconstitute putative signaling pathways in vitro with purified components to demonstrate direct phosphorylation

Temporal analysis using time-course experiments after stimulus application provides crucial insights into the signaling cascade sequence leading to catalase phosphorylation .

How does catalase phosphorylation status change in pathological conditions and what are the methodological considerations for such analyses?

Catalase phosphorylation status may be altered in various pathological conditions, particularly those involving oxidative stress or dysregulated signaling pathways. Key methodological considerations for studying these changes include:

• Sample preservation: Rapid sample collection and processing are critical as phosphorylation states can change quickly post-mortem or ex vivo

• Phosphatase inhibition: Include comprehensive phosphatase inhibitor cocktails in all buffers to prevent artificial dephosphorylation during sample handling

• Normalization approaches: Use multiple housekeeping proteins for normalization, as some traditional controls may be altered in disease states

• Tissue heterogeneity: Consider cell type-specific changes through techniques like laser capture microdissection or single-cell analysis

• Appropriate controls: Include age-matched, sex-matched controls and consider medication effects in human samples

• Cross-validation: Confirm phosphorylation changes using multiple techniques (Western blot, immunohistochemistry, mass spectrometry)

Disease conditions where catalase phosphorylation may be particularly relevant include:

• Neurodegenerative disorders (Alzheimer's, Parkinson's)

• Cancer (various types)

• Cardiovascular diseases (atherosclerosis, hypertension)

• Metabolic disorders (diabetes, obesity)

• Inflammatory conditions (arthritis, inflammatory bowel disease) .

What are the common sources of false positive and false negative results when using Phospho-CAT (Y386) Antibody?

When using Phospho-CAT (Y386) Antibody, researchers should be aware of potential sources of false results:

False Positive Sources:

• Cross-reactivity with similar phospho-epitopes in other proteins

• Non-specific binding to denatured proteins in over-fixed samples

• Insufficient blocking leading to high background

• Secondary antibody cross-reactivity

• Spontaneous phosphorylation during sample processing

False Negative Sources:

• Phosphatase activity during sample preparation

• Epitope masking due to protein-protein interactions

• Suboptimal fixation conditions destroying the phospho-epitope

• Insufficient antibody concentration or incubation time

• Low abundance of the phosphorylated form of catalase

Quality Control Measures:

• Include positive controls (in vitro phosphorylated samples)

• Include negative controls (phosphatase-treated samples)

• Validate results with alternative detection methods

• Perform peptide competition assays to confirm specificity

• Use phospho-null mutants (Y386F) as additional controls .

How can researchers optimize antibody concentration and incubation conditions for maximum specificity and sensitivity?

Optimizing antibody concentration and incubation conditions is critical for achieving maximum specificity and sensitivity with Phospho-CAT (Y386) Antibody:

Antibody Dilution Optimization:

• Perform a dilution series (e.g., 1:100, 1:500, 1:1000, 1:2000, 1:5000)

• For Western blot, start with the recommended range (1:500-1:2000)

• For IHC, begin with the suggested range (1:100-1:300)

• For ELISA, use higher dilutions as recommended (1:20000)

• Select the concentration that provides the best signal-to-noise ratio

Incubation Conditions Optimization:

• Temperature variations: Test 4°C overnight, room temperature (4 hours), and 37°C (1 hour)

• Buffer composition: Optimize blocking agents (BSA, milk, serum) and detergent concentration

• Duration: Test different incubation times to find optimal signal development

• Sequential incubation: Consider sequential rather than simultaneous incubation for multiple antibodies

Methodological Approach to Optimization:

• Use a matrix-based approach testing multiple parameters simultaneously

• Keep all other variables constant when testing a specific parameter

• Document all conditions meticulously

• Validate optimal conditions across different sample types

• Perform replicate experiments to ensure reproducibility .

What validation experiments should be performed to confirm the specificity of Phospho-CAT (Y386) Antibody before proceeding with experimental applications?

Before using Phospho-CAT (Y386) Antibody in experimental applications, researchers should perform a series of validation experiments to confirm specificity:

• Phosphatase Treatment:

  • Treat duplicate samples with lambda phosphatase

  • Compare treated vs. untreated samples by Western blot

  • Signal should disappear in phosphatase-treated samples

• Peptide Competition Assay:

  • Pre-incubate antibody with excess phosphorylated and non-phosphorylated peptides

  • Signal should be blocked by phosphorylated but not non-phosphorylated peptide

• Phospho-null Mutant Analysis:

  • Express wild-type catalase and Y386F mutant

  • Antibody should detect wild-type but not Y386F mutant

• Mass Spectrometry Correlation:

  • Confirm phosphorylation at Y386 by mass spectrometry

  • Compare antibody reactivity pattern with MS results

• Kinase/Phosphatase Modulation:

  • Treat cells with kinase activators/inhibitors

  • Observe corresponding changes in antibody signal intensity

• Cross-reactivity Testing:

  • Test antibody against closely related phosphorylation sites

  • Ensure signal specificity to the Y386 site

• Knockout/Knockdown Validation:

  • Use catalase knockout/knockdown cells or tissues

  • Confirm absence of signal in these samples

Proper validation not only ensures experimental reliability but also provides crucial controls for future experiments and troubleshooting .