ARPP19 Antibody

What is ARPP19 and why is it significant for cell cycle research?

ARPP19 (cAMP-regulated phosphoprotein 19 kD) is a critical protein phosphatase inhibitor that specifically inhibits protein phosphatase 2A (PP2A) during mitosis. When phosphorylated at Ser-62 during mitosis, ARPP19 specifically interacts with PPP2R2D (PR55-delta) and inhibits its activity, leading to inactivation of PP2A, which is essential for maintaining high cyclin-B1-CDK1 activity during M phase . ARPP19 plays a crucial role in:

• Regulating mitotic progression

• Controlling meiotic resumption in oocytes

• Embryonic development through cell cycle regulation

• Maintaining proper chromosome alignment and segregation

Research has shown that ARPP19 knockout in mice results in embryonic lethality, with embryos showing severe abnormalities by E8.5 and failed gastrulation, demonstrating its essential role in early development .

What applications are ARPP19 antibodies validated for in research settings?

ARPP19 antibodies have been validated for multiple research applications across different experimental platforms:

It's important to note that experimental conditions may need optimization depending on your specific cell type or tissue of interest .

How should ARPP19 antibodies be stored and handled to maintain optimal activity?

Based on manufacturer recommendations for ARPP19 antibodies, optimal storage and handling practices include:

• Store at -20°C for long-term preservation; stable for one year after shipment

• For short-term storage and frequent use, keep at 4°C for up to one month

• Many ARPP19 antibodies are supplied in storage buffer containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Avoid repeated freeze-thaw cycles as this can diminish antibody performance

• Some antibody preparations (20μl sizes) may contain 0.1% BSA as a stabilizer

• Aliquoting is generally unnecessary for -20°C storage

These handling practices are critical for maintaining antibody specificity and performance across experimental applications.

How do you select between antibodies targeting total ARPP19 versus phospho-specific forms?

Selection between total and phospho-specific ARPP19 antibodies depends on your research question:

For Total ARPP19 Detection:

• Use when measuring expression levels across different tissues or cell types

• Suitable for immunoprecipitation experiments before analyzing phosphorylation status

• Appropriate for detecting knockout/knockdown efficiency

• Can be used to normalize phosphorylation signals in quantitative studies

For Phospho-Specific Detection:

• Use phospho-S109 antibodies when studying PKA-mediated inhibition of meiotic maturation, as this phosphorylation is essential for maintaining oocyte prophase arrest

• Use phospho-S67 antibodies when investigating mitotic regulation, as this modification by Greatwall kinase is critical for PP2A inhibition during M-phase

• Phospho-antibodies require careful sample preparation to preserve phosphorylation states

• Consider using phosphatase inhibitors during sample preparation

Research has demonstrated that ARPP19 phosphorylation state changes dynamically during cell cycle progression, with S109 dephosphorylation occurring within 60 minutes following progesterone treatment of Xenopus oocytes (49% reduction) .

What controls should be included when validating ARPP19 antibody specificity?

Rigorous validation of ARPP19 antibodies should include:

Positive Controls:

• Known positive cell lines: Jurkat, A375, and A549 cells express detectable levels of ARPP19

• Tissue samples: Mouse brain and rat skeletal muscle tissue have validated ARPP19 expression

• Recombinant ARPP19 protein: Can serve as a positive control and calibration standard

Negative Controls:

• ARPP19 knockout/knockdown samples: Use Arpp19 Δ/Δ MEFs or CRISPR/Cas9 edited cell lines

• Blocking peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

• Secondary-only controls: To detect non-specific binding of secondary antibodies

Specificity Verification:

• Cross-reactivity testing: Confirm your antibody doesn't detect the closely related ENSA protein

• Immunoblotting should show a band at the expected molecular weight (19 kDa)

• For phospho-specific antibodies, treatment with lambda phosphatase should eliminate the signal

Research has shown that reliable detection of endogenous ARPP19 may require immunoprecipitation before Western blotting due to its low abundance (approximately 0.06 ng/μg total protein in MEFs) .

How can ARPP19 antibodies be used to study mitotic defects in knockout or knockdown models?

ARPP19 antibodies are valuable tools for investigating mitotic defects in knockout/knockdown models:

Experimental Approach:

• Generate ARPP19-depleted cells (using CRISPR/Cas9, shRNA, or conditional knockout)

• Assess mitotic progression using immunofluorescence microscopy with:

  • Anti-ARPP19 to confirm depletion

  • Anti-phospho-histone H3 to identify mitotic cells

  • Anti-BUBR1 to evaluate spindle assembly checkpoint activation

  • DNA staining to assess chromosome condensation and segregation

Expected Phenotypes Based on Research:

• Arpp19 Δ/Δ MEFs exhibit multiple mitotic defects including:

  • Chromosome misalignment (3.5× more frequent than controls)

  • Missegregation and DNA bridges (50% higher than controls)

  • Micronuclei formation (28% vs. 3% in controls)

  • Multinucleated cells (13% vs. 3% in controls)

  • Chromosome decondensation (16% vs. 0.6% in controls)

Rescue Experiments:

• Express wild-type ARPP19 or phosphorylation site mutants (S62A)

• Monitor restoration of normal mitotic progression

• Data shows that S62A mutant cannot rescue viability in ARPP19-depleted cells

When designing these experiments, it's important to note that ARPP19 depletion does not affect spindle assembly checkpoint activation, as evidenced by normal nocodazole-induced mitotic arrest and increased BUBR1 signal at kinetochores .

What are the methodological considerations when using phospho-specific ARPP19 antibodies?

When working with phospho-specific ARPP19 antibodies, several methodological considerations are critical:

Sample Preparation:

• Use phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

• Process samples quickly at 4°C to prevent dephosphorylation

• For cross-linking studies examining ARPP19-PP2A interactions, use reversible cross-linkers in lysis buffers containing DTT and EDTA (without Mg²⁺) to stabilize complexes while inactivating endogenous kinases

Antibody Selection:

• For S109 phosphorylation (PKA site): Use anti-phospho-S109 antibodies that recognize the sequence CQDLPQRKPpSLVASK

• For S67 phosphorylation (Greatwall site): Use anti-phospho-S67 antibodies specific to this regulatory site

Experimental Design:

• Include positive controls: Progesterone treatment of Xenopus oocytes causes S109 dephosphorylation (49% reduction within 60 minutes)

• Include negative controls: Non-phosphorylatable mutants (S109A or S67A) should not be recognized by the respective phospho-antibodies

• Consider time-course experiments: ARPP19 phosphorylation states change dynamically during cell cycle progression

Signal Detection:

• Due to low endogenous levels, immunoprecipitation before Western blotting may be necessary

• In Xenopus oocytes, ARPP19 is progressively rephosphorylated at S109 after initial dephosphorylation, reaching or exceeding prophase levels at GVBD

These methodological considerations are essential for obtaining reliable and interpretable results when studying the complex regulation of ARPP19 phosphorylation.

How can researchers troubleshoot weak or non-specific signals when using ARPP19 antibodies?

When encountering challenges with ARPP19 antibody signals, systematic troubleshooting approaches include:

For Weak Signals:

• Increase protein loading: Endogenous ARPP19 is often expressed at low levels (0.06 ng/μg total protein in MEFs)

• Optimize antibody concentration: Test a range of dilutions around the recommended dilution

• Enhance detection methods:

  • For Western blots: Use Tris-Tricine gels (15.5%) instead of standard Tris-Glycine for better resolution of low molecular weight proteins

  • Load higher amounts (equivalent of 1.5 oocytes vs. standard 0.5) for endogenous detection

• Consider immunoprecipitation: Enrich ARPP19 before detection - research shows endogenous ARPP19 is "hardly detectable by Western blotting, but clearly visible when immunoprecipitated"

• Optimize blocking conditions: Test different blocking agents (BSA vs. milk)

For Non-specific Signals:

• Cross-reactivity testing: Verify the antibody doesn't detect closely related proteins, particularly ENSA

• Validation with knockout samples: Use Arpp19 Δ/Δ cells as negative controls

• Optimize washing conditions: Increase wash duration or detergent concentration

• Use freshly prepared samples: Degraded samples can increase background

• Test different antibody batches: Quality can vary between lots

Application-Specific Solutions:

• For IHC applications: Test different antigen retrieval methods - TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

• For IF applications: Optimize fixation methods and permeabilization conditions

• For phospho-specific antibodies: Ensure phosphatase inhibitors are included in all buffers

Implementing these troubleshooting approaches systematically can significantly improve both specificity and sensitivity when working with ARPP19 antibodies.

How do ARPP19 mutations affect antibody selection for studying its role in cell cycle regulation?

Different ARPP19 mutations require strategic antibody selection to effectively study their impact on cell cycle regulation:

Phosphorylation Site Mutants:

• S62A/S67A mutants (non-phosphorylatable at the Greatwall site):

  • Cannot be detected by phospho-S62/S67 antibodies

  • Use total ARPP19 antibodies to confirm expression

  • Research shows these mutants fail to rescue viability in ARPP19-depleted cells, confirming the essential nature of this phosphorylation

• S109A/D mutants (affecting the PKA site):

  • S109D phosphomimetic mutant blocks progesterone- and PKI-induced prophase release

  • Use total ARPP19 antibodies to track expression levels

  • Monitor downstream effects using antibodies against phosphorylated MAPK and Cdk substrates

Binding Site Mutants:

• G72A mutant (increased PP2A-B55 affinity):

  • Accelerates oocyte maturation compared to wild-type ARPP19

  • Use both total and phospho-specific antibodies to track expression and modification

  • Monitor dephosphorylation kinetics using autoradiography and western blotting

• D70A mutant (rapid dephosphorylation of S67/S71):

  • Modified dephosphorylation kinetics make this mutant valuable for temporal studies

  • Use time-course experiments with phospho-specific antibodies

  • Combine with PP2A antibodies to study interaction dynamics

Experimental Strategy:
For studying mutant effects on cell cycle, combine:

• Total ARPP19 antibodies to confirm expression levels

• Phospho-specific antibodies to track modification states

• Cell cycle markers (phospho-histone H3, cyclins, Cdk substrates) to monitor progression

• PP2A-B55 antibodies to assess interactions

This comprehensive approach enables mechanistic insights into how specific ARPP19 mutations affect its regulation of the cell cycle.

What are the species-specific considerations when selecting ARPP19 antibodies for comparative studies?

When conducting comparative studies across species, careful antibody selection is essential:

Species Reactivity Profiles:
The following table summarizes documented species reactivity for commercial ARPP19 antibodies:

Sequence Homology Considerations:

• Human and mouse ARPP19 share high sequence homology, but epitope recognition should be confirmed experimentally

• Epitope location is critical - the antibody against ARPP19 N-terminus (as described in ) was specifically designed not to cross-react with the related protein ENSA

• For phospho-specific antibodies, verify conservation of the phosphorylation site across species

Validation Approaches:

• Perform Western blot analysis on samples from each species of interest

• Use species-specific positive controls (e.g., mouse brain tissue for mouse studies)

• Consider using recombinant His-ARPP19 as a calibration standard

• For knockout validation, Arpp19 Δ/Δ MEFs provide an excellent negative control for mouse studies

Cross-Reactivity Testing:

• Test for cross-reactivity with ENSA, which shares significant homology with ARPP19

• Verify specificity using knockout samples from each species when available

• For phospho-antibodies, consider species differences in kinase recognition motifs

These considerations ensure valid cross-species comparisons when studying ARPP19 function in evolutionary and comparative biology contexts.

How can ARPP19 antibodies be used to study its interaction with PP2A during cell cycle regulation?

To study ARPP19-PP2A interactions during cell cycle progression:

Co-Immunoprecipitation Protocol:

• Prepare cell lysates in a buffer containing reversible cross-linker to stabilize PP2A-ARPP19 complexes

• Use conditions that inactivate endogenous kinases (DTT, EDTA, without Mg²⁺)

• Immunoprecipitate using anti-ARPP19 antibodies

• Analyze precipitates by Western blotting with:

  • Anti-PP2A catalytic subunit antibodies

  • Anti-PP2A regulatory subunit antibodies (especially B55δ)

  • Anti-phospho-S67/S62 ARPP19 antibodies

Temporal Analysis During Cell Cycle:

• Synchronize cells at different cell cycle stages using standard protocols

• Perform co-IP at defined timepoints to track dynamic interactions

• Analyze phosphorylation status of ARPP19 concurrent with PP2A binding

Mutant Studies:

• Compare wild-type ARPP19 with:

  • S67A (non-phosphorylatable Greatwall site)

  • G72A (increased PP2A-B55 affinity mutant)

  • D70A (rapid dephosphorylation mutant)

Competition Assays:

• Use increasing amounts of wild-type GST-ARPP to study dose-dependent effects on meiotic resumption

• Research shows high amounts of WT-GST-ARPP block meiotic resumption in a dose-dependent manner

These approaches provide mechanistic insights into how ARPP19 phosphorylation regulates its interaction with PP2A during cell cycle transitions.

What quantitative methods are recommended for analyzing ARPP19 phosphorylation dynamics?

For quantitative analysis of ARPP19 phosphorylation dynamics:

Quantitative Western Blotting:

• Use recombinant His-ARPP19 as a calibration standard

• Create a standard curve with known amounts (research shows endogenous ARPP19 is approximately 0.06 ng/μg total protein in MEFs)

• Normalize phospho-signals to total ARPP19 levels

• Use digital imaging systems with linear detection range

• Apply appropriate statistical analysis to replicate experiments

Phosphorylation Kinetics Assessment:

• For S109 dephosphorylation: Research shows progesterone treatment of Xenopus oocytes causes 49% reduction (s.d.=19, n=4) within 60 minutes

• For re-phosphorylation dynamics: Track progressive rephosphorylation reaching prophase levels at GVBD

• For S67/S71 dephosphorylation: Use autoradiography of in vitro phosphorylated mutants in kinase-inactivated egg extracts

Time-Course Analysis Example:
Measure relative ARPP19 phosphorylation at different timepoints after treatment:

Imaging-Based Quantification:

• For single-cell analysis, use quantitative immunofluorescence

• Normalize to appropriate housekeeping proteins

• Consider advanced approaches like FRET-based sensors for real-time dynamics

These quantitative approaches enable precise measurement of ARPP19 phosphorylation dynamics during cell cycle transitions and in response to experimental manipulations.

What are the recommended experimental controls when using ARPP19 antibodies for developmental studies?

When using ARPP19 antibodies in developmental research, comprehensive controls are essential:

Genotype Verification Controls:

• For Arpp19 knockout studies: Confirm genotype using PCR and validate protein absence using Western blotting with ARPP19 antibodies

• For conditional knockouts: Verify Cre-mediated excision efficiency in specific tissues

Developmental Stage-Specific Controls:

• Use stage-matched wild-type embryos alongside experimental samples

• In mice, embryonic lethality occurs by E8.5 in Arpp19 Δ/Δ embryos, with 100% showing severe abnormalities

• For cell type-specific analyses, include differentiation markers appropriate to developmental stage

Functional Rescue Controls:

• Test rescue with wild-type ARPP19 versus phosphorylation site mutants

• Research shows S62A mutant fails to rescue viability in ARPP19-depleted cells

• Include appropriate vector-only controls

Expression Pattern Controls:

• Use Arpp19 gene reporter mice (e.g., lacZ-trapping element) to determine expression patterns during embryogenesis

• Validate reporter expression with antibody staining when possible

Tissue-Specific Controls:

• For immunohistochemistry: Include positive control tissues (human adrenal gland and placenta have been validated)

• For mitotic analysis in embryonic tissue: Use anti-phosphorylated histone H3 antibody to identify mitotic cells

• Research shows significant increase in mitotic cells in the epidermal basal layer of Arpp19-depleted embryos

Technical Controls:

• Include secondary-only controls for all immunostaining

• For phospho-antibodies: Include lambda phosphatase-treated samples as negative controls

• Use isotype controls to control for non-specific binding

These comprehensive controls ensure reliable interpretation of ARPP19 functions in developmental contexts.

How should researchers interpret discrepancies between different ARPP19 antibody detection methods?

When encountering discrepancies between different detection methods:

Common Discrepancies and Interpretation:

• Discrepancy between Western blot and immunostaining:

  • Western blot detects denatured epitopes while immunostaining requires native conformation

  • ARPP19 is "hardly detectable by Western blotting, but clearly visible when immunoprecipitated"

  • Solution: Verify antibody compatibility with each method and optimize protocol accordingly

• Variations between phospho-specific and total antibody signals:

  • Consider rapid phosphorylation/dephosphorylation dynamics

  • Phosphatase activity during sample preparation can reduce phospho-signals

  • In Xenopus oocytes, S109 dephosphorylation occurs within 60 minutes (49% reduction) followed by rephosphorylation

  • Solution: Use phosphatase inhibitors and rapid sample processing

• Discrepancy between endogenous versus overexpressed ARPP19:

  • Endogenous levels are low (~0.06 ng/μg total protein in MEFs)

  • Overexpression may alter stoichiometry of interactions and phosphorylation

  • Solution: Quantify expression levels and compare to endogenous references

• Inconsistent results across species:

  • Confirm antibody cross-reactivity with the species being studied

  • Consider epitope conservation across species

  • Solution: Validate each antibody with species-specific positive controls

Reconciliation Approach:

• Validate antibody in each application separately

• Use multiple antibodies targeting different epitopes

• Include appropriate positive and negative controls

• Complement antibody-based detection with other methods (mass spectrometry, activity assays)

• Consider the biological context of each experimental system

This systematic approach helps resolve discrepancies and ensures reliable interpretation of ARPP19 data across different detection methods.

What are the critical considerations for quantifying ARPP19 levels in samples with low expression?

For accurate quantification of ARPP19 in low-expression samples:

Sample Enrichment Strategies:

• Immunoprecipitation before Western blotting:

  • Research shows endogenous ARPP19 is "hardly detectable by Western blotting, but clearly visible when immunoprecipitated"

  • Use specific ARPP19 N-terminal antibodies that don't cross-react with ENSA

  • Include DTT and EDTA in lysis buffer when studying interactions

• Subcellular fractionation:

  • Concentrate ARPP19 by isolating relevant cellular compartments

  • Nuclear/cytoplasmic fractionation can increase detection sensitivity

Optimized Detection Methods:

• Specialized gel systems:

  • Use 15.5% Tris-Tricine gels instead of standard Tris-Glycine for better resolution of low molecular weight proteins

  • Load higher sample amounts (equivalent of 1.5 oocytes vs. standard 0.5) for endogenous detection

• Enhanced chemiluminescence:

  • Use high-sensitivity substrates for Western blotting

  • Optimize exposure times for weak signals without saturating standards

Quantification Approaches:

• Standard curve calibration:

  • Use recombinant His-ARPP19 as calibration standard

  • Create standard curves with concentrations spanning expected sample range

• Internal controls:

  • Include loading controls appropriate for the sample type

  • Normalize to housekeeping proteins with similar abundance to ARPP19

• Digital imaging analysis:

  • Use systems with high dynamic range and sensitivity

  • Apply appropriate background subtraction methods

  • Consider analyzing multiple exposure times to ensure signals are in linear range

Technical Considerations:

• Avoid membrane stripping which can reduce sensitivity

• Use PVDF membranes which typically offer better protein retention than nitrocellulose

• Consider fluorescent secondary antibodies for more precise quantification

• Account for sample-to-sample variability by analyzing biological replicates

These approaches enable reliable quantification of ARPP19 even in samples with naturally low expression levels.

How can researchers distinguish between ARPP19 and ENSA in experimental systems?

Distinguishing ARPP19 from its close homolog ENSA is critical for accurate experimental interpretation:

Antibody Selection Strategy:

• Use antibodies specifically designed not to cross-react with ENSA, such as those targeting the N-terminus of ARPP19

• Validate antibody specificity using overexpression and knockout controls for both ARPP19 and ENSA

• Consider using epitope-tagged versions when studying overexpressed proteins

Molecular Weight Discrimination:

• ARPP19: 19 kDa observed molecular weight

• ENSA: 13-15 kDa (depending on the isoform)

• Use high-resolution gel systems (15.5% Tris-Tricine gels) for optimal separation

Genetic Approaches:

• Use specific siRNA/shRNA targeting unique regions of each transcript

• Employ Arpp19 Δ/Δ MEFs as a model system lacking ARPP19 but retaining ENSA

• Create CRISPR/Cas9 knockout models for each protein separately

Functional Discrimination:

Expression Pattern Analysis:

• Use Arpp19 gene reporter mice to determine specific expression patterns during embryogenesis

• Compare with ENSA expression patterns in the same tissues

• Combine with immunohistochemistry using specific antibodies