PPP2R3A Antibody, HRP conjugated

What is PPP2R3A and why is it significant in research?

PPP2R3A (Protein Phosphatase 2, Regulatory Subunit B'', alpha) is a regulatory subunit of the protein phosphatase 2A (PP2A) complex. This protein is significant in research because the B regulatory subunit modulates substrate selectivity and catalytic activity while also directing the localization of the catalytic enzyme to particular subcellular compartments . Recent studies have revealed that PPP2R3A plays critical roles in hepatocellular carcinoma (HCC) development by regulating glycolysis through targeting hexokinase 1 (HK1) . Understanding PPP2R3A function provides insights into cellular signaling pathways involved in cancer development and progression.

What are the key characteristics of PPP2R3A antibodies?

PPP2R3A antibodies are typically characterized by:

• Target Specificity: Recognizes specific amino acid regions (e.g., AA 256-508) of the PPP2R3A protein

• Host Species: Commonly raised in rabbits for polyclonal antibodies

• Clonality: Available in polyclonal formats

• Reactivity: Primary reactivity with human samples, though some variants may cross-react with mouse and rat samples

• Applications: Commonly used in ELISA, immunohistochemistry (IHC), and sometimes Western blotting

• Conjugation: Available in both unconjugated and conjugated forms (HRP, FITC)

What experimental applications are suitable for HRP-conjugated PPP2R3A antibodies?

HRP-conjugated PPP2R3A antibodies are particularly suitable for:

• ELISA: The HRP conjugation provides direct enzymatic detection without requiring secondary antibodies, enabling sensitive quantification of PPP2R3A in complex samples

• Immunohistochemistry: Can be used for direct detection in tissue sections, though primary applications focus on ELISA

• Colorimetric detection systems: The HRP enzyme catalyzes chromogenic reactions for visualization in multiple platforms

• Western blotting: Although not explicitly mentioned for the HRP-conjugated version, the antibody characteristics suggest compatibility with this application when properly optimized

How should researchers optimize PPP2R3A antibody dilutions for different applications?

Optimization of PPP2R3A antibody dilutions is critical for experimental success:

• Initial titration: Begin with manufacturer-recommended dilutions (information not explicitly provided in search results but typically ranges from 1:500 to 1:2000 for HRP-conjugated antibodies)

• ELISA optimization:

  • Start with a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:4000)

  • Select the dilution providing optimal signal-to-noise ratio

  • Consider the buffer composition (the antibody is supplied in preservative containing 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4)

• Immunohistochemistry considerations:

  • Typically requires lower dilutions than ELISA

  • Include appropriate positive and negative controls

  • Consider antigen retrieval requirements

• Validation: Compare results across multiple applications to ensure consistency and specificity

What storage conditions are optimal for maintaining PPP2R3A antibody activity?

To maintain optimal activity of HRP-conjugated PPP2R3A antibodies:

• Storage temperature: Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles: These can significantly degrade antibody activity and HRP conjugation

• Working aliquots: Prepare small aliquots for routine use to minimize freeze-thaw cycles

• Buffer considerations: The antibody is supplied in 50% glycerol buffer, which helps maintain stability during freezing

• Shipping conditions: When receiving the antibody, ensure it was shipped properly on ice or dry ice

• Monitoring: Regularly verify activity by including appropriate positive controls in experiments

How can PPP2R3A antibodies be used to investigate the relationship between PPP2R3A and HK1 in cancer research?

Recent research has revealed a significant relationship between PPP2R3A and hexokinase 1 (HK1) in hepatocellular carcinoma:

• Co-localization studies:

  • Dual immunofluorescence staining has demonstrated that PPP2R3A and HK1 are co-localized in the cytoplasm of HCC cells

  • Strong positive correlation (r = 0.906, P = 0.001) between PPP2R3A and HK1 protein expression levels in primary HCC samples

• Functional relationship analysis:

  • PPP2R3A overexpression increases HK1 gene and protein expression in HepG2 and Huh7 liver cancer cell lines

  • PPP2R3A knockdown reduces HK1 expression levels

  • Database analysis (GEPIA and TCGA) confirms a positive correlation between PPP2R3A and HK1 expression in HCC samples (r = 0.28 and r = 0.26, respectively)

• Glycolysis pathway investigation:

  • PPP2R3A overexpression increases glucose uptake and lactate production in liver cancer cells

  • These effects are mediated through HK1, the first rate-limiting enzyme in the glycolytic pathway

What are potential confounding factors when using PPP2R3A antibodies in research, and how can they be addressed?

Several factors can confound results when using PPP2R3A antibodies:

• Antibody specificity issues:

  • Verify specificity through multiple techniques (Western blot, IHC, IF)

  • Include appropriate positive and negative controls

  • Consider using knockdown/knockout samples as negative controls

• Cross-reactivity considerations:

  • Some PPP2R3A antibodies may cross-react with mouse and rat samples

  • Validate specificity when working with non-human samples

  • Select antibodies with species-specific validation

• Signal detection challenges:

  • HRP conjugation may lead to nonspecific background in certain tissues

  • Optimize blocking conditions (typically 5% BSA or non-fat milk)

  • Include appropriate enzyme inhibitors when working with tissues with high endogenous peroxidase activity

• Functional redundancy in the PP2A complex:

  • PPP2R3A is one of several regulatory B subunits

  • Consider parallel analysis of related PP2A subunits to address functional redundancy

  • Validate findings through multiple approaches (genetic manipulation, pharmacological inhibition)

What is the optimal protocol for using PPP2R3A antibody in dual immunofluorescence studies?

Based on successful application in recent research , the following protocol is recommended for dual immunofluorescence studies:

• Sample preparation:

  • Fix tissue samples in 4% paraformaldehyde

  • Process and embed in paraffin or optimal cutting temperature compound for frozen sections

  • Cut sections at 4-6 μm thickness

• Antigen retrieval:

  • Deparaffinize and rehydrate sections if using paraffin-embedded tissues

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Allow slides to cool to room temperature

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS containing 0.1% Triton X-100 for 1 hour

  • Incubate with rabbit anti-PPP2R3A antibody (optimized dilution) overnight at 4°C

  • Wash 3× with PBS

  • Incubate with mouse anti-HK1 antibody for 2 hours at room temperature

  • Wash 3× with PBS

• Secondary antibody incubation:

  • Use species-specific secondary antibodies with different fluorophores

    • Anti-rabbit secondary antibody (red fluorescence)

    • Anti-mouse secondary antibody (green fluorescence)

  • Include DAPI for nuclear counterstaining

  • Control for cross-reactivity between secondary antibodies

• Analysis:

  • Examine colocalization using confocal microscopy

  • Quantify mean fluorescence intensity for correlation analysis

  • Analyze Pearson's correlation coefficient to determine relationship strength

How can researchers validate the specificity of PPP2R3A antibody for their experimental system?

A comprehensive validation approach includes:

• Multiple antibody comparison:

  • Test antibodies targeting different epitopes of PPP2R3A

  • Compare polyclonal and monoclonal antibodies when available

  • Include antibodies from different vendors

• Genetic validation:

  • Use PPP2R3A knockdown (siRNA) or knockout samples as negative controls

  • Perform rescue experiments by reintroducing PPP2R3A expression

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare signal with and without competing peptide

• Cross-platform validation:

  • Confirm findings using multiple techniques (Western blot, ELISA, IHC, IF)

  • Ensure consistent results across different detection methods

• Expression pattern analysis:

  • Verify that staining pattern matches known subcellular localization (cytoplasmic for PPP2R3A)

  • Compare with published literature on PPP2R3A localization

How should researchers interpret PPP2R3A and HK1 correlation data in the context of cancer research?

The correlation between PPP2R3A and HK1 in cancer research should be interpreted with the following considerations:

• Statistical correlation strength:

  • Strong positive correlation in primary HCC samples (r = 0.906, P = 0.001)

  • Moderate positive correlation in database analyses (GEPIA: r = 0.28, P < 0.05; TCGA: r = 0.26, P < 0.05)

  • Consider sample size and heterogeneity when interpreting correlation coefficients

• Functional relationship:

  • PPP2R3A positively regulates HK1 expression at both gene and protein levels

  • This regulation appears to be directional (PPP2R3A → HK1) based on knockdown and overexpression studies

  • The relationship impacts glycolysis, a key metabolic pathway in cancer cells

• Biological significance:

  • HK1 is the first rate-limiting enzyme in glycolysis

  • The PPP2R3A-HK1 axis affects glucose uptake and lactate production in cancer cells

  • These metabolic changes may contribute to the oncogenic roles of PPP2R3A

• Clinical implications:

  • Consider potential as biomarkers or therapeutic targets

  • Evaluate relationship in multiple cancer types beyond HCC

  • Assess correlation with clinical outcomes and disease progression

What challenges exist in reconciling in vitro findings with clinical data when using PPP2R3A antibodies?

Several challenges must be addressed when reconciling laboratory and clinical findings:

• Antibody performance differences:

  • Fixation methods in clinical samples may affect epitope accessibility

  • Background staining patterns may differ between cell lines and tissues

  • Standardization of staining protocols is critical for comparison

• Heterogeneity considerations:

  • Cancer tissue contains multiple cell types beyond cancer cells

  • Regional heterogeneity within tumors may affect PPP2R3A expression patterns

  • Consider microdissection or single-cell approaches for precise analysis

• Pathway complexity:

  • PPP2R3A functions within the complex PP2A regulatory network

  • Multiple upstream regulators and downstream effectors exist

  • In vitro models may not capture the full complexity of pathway interactions

• Translation of functional findings:

  • Metabolic changes (glycolysis) observed in cell lines may be influenced by culture conditions

  • The tumor microenvironment affects cancer metabolism in ways not replicated in vitro

  • Consider complementary approaches (e.g., patient-derived xenografts, ex vivo tissue culture)