PPP1R2 (Ab-120/121) Antibody

What is PPP1R2 and what cellular functions does it regulate?

PPP1R2 (Protein Phosphatase 1 Regulatory Inhibitor Subunit 2) is a heat-stable inhibitor of protein phosphatase 1 (PP1), an essential serine/threonine phosphatase involved in numerous cellular processes. PPP1R2 inhibits PP1 in its dephosphorylated form, unlike some other inhibitors which require phosphorylation for activity . The protein plays crucial roles in cell cycle regulation, cell division processes, and cell proliferation pathways.

PPP1R2 is part of an evolutionarily ancient and highly conserved family of phosphatase regulators that shows significant expression in testicular tissue and developing spermatogenic cells . The inhibitor interacts with glycogen synthase kinase 3 (GSK3), which can phosphorylate PPP1R2 and reverse its inhibitory effect on PP1 . Dysregulation of PPP1R2 has been associated with various diseases, including cancer and developmental disorders .

How does the PPP1R2 (Ab-120/121) Antibody differ from other PPP1R2 antibodies?

The PPP1R2 (Ab-120/121) Antibody is specifically designed to recognize the region surrounding phosphorylation sites at serine 120 and serine 121 of human PPP1R2. This distinguishes it from other antibodies in several important ways:

• It targets a specific phosphorylation-sensitive epitope with the sequence Q-E-S(p)-S(p)-G-E

• It was generated using a synthesized non-phosphopeptide derived from human PPP1R2

• It can potentially distinguish between different phosphorylation states at these specific regulatory sites

• It demonstrates cross-reactivity with human, mouse, and rat PPP1R2

• It has been affinity-purified from rabbit antiserum using epitope-specific immunogen

This specificity makes it particularly valuable for studies examining the phosphorylation status of PPP1R2 at these sites, which may affect its inhibitory activity toward PP1.

What applications is the PPP1R2 (Ab-120/121) Antibody validated for?

The PPP1R2 (Ab-120/121) Antibody has been validated for multiple research applications:

The antibody has been specifically tested on human breast carcinoma tissue samples for IHC applications and in cell extracts from JK and Jurkat cells for Western blotting . Visual validation of the antibody's performance in Western blot analysis shows clear detection of PPP1R2 in Jurkat cell extracts, with specificity confirmed through peptide competition controls . The antibody has demonstrated reliable performance in detecting endogenous levels of PPP1R2 protein across multiple experimental systems.

What is the specificity of PPP1R2 (Ab-120/121) Antibody across different species?

The PPP1R2 (Ab-120/121) Antibody demonstrates cross-reactivity with multiple species:

This cross-species reactivity is due to the high conservation of the epitope region across mammalian species . The antibody was generated against a synthetic peptide derived from human PPP1R2 around the phosphorylation sites of serine 120 and serine 121. The high degree of conservation in this region allows for broader species application, making it valuable for comparative studies in different model organisms. Researchers have successfully used this antibody to detect PPP1R2 in various tissues from these species, including testicular and sperm samples where PPP1R2 shows notable expression .

What is the immunogen used to generate the PPP1R2 (Ab-120/121) Antibody?

The PPP1R2 (Ab-120/121) Antibody was generated using a synthesized non-phosphopeptide derived from human PPP1R2 specifically targeting the region around the phosphorylation sites of serine 120 and serine 121. The precise immunogen sequence is Q-E-S(p)-S(p)-G-E .

This strategic choice of immunogen provides several research advantages:

• It targets a functionally relevant region involved in regulation of PPP1R2 activity

• It may allow detection of different phosphorylation states of the protein

• The region is highly conserved, allowing cross-species application

• It permits monitoring of post-translational modifications that regulate PPP1R2 function

The antibody was produced in rabbits and subsequently purified using affinity chromatography with the epitope-specific immunogen to ensure high specificity .

How should optimization of Western blotting conditions be approached when using PPP1R2 (Ab-120/121) Antibody?

Optimizing Western blotting with the PPP1R2 (Ab-120/121) Antibody requires systematic adjustment of several parameters:

• Sample Preparation and Loading:

  • Use fresh samples with protease and phosphatase inhibitors to preserve phosphorylation states

  • Include positive controls (e.g., Jurkat or JK cell extracts, as validated in product data)

  • Include a peptide competition control with the synthesized peptide

  • Load 20-50 μg of total protein per lane for cell lysates

• Primary Antibody Dilution Optimization:

  • Begin with a 1:1000 dilution (within the recommended 1:500-1:3000 range)

  • Prepare a dilution series if signal intensity is unknown

  • Dilute in blocking buffer containing 0.05% Tween-20

• Incubation Conditions:

  • Optimize between 4°C overnight incubation or 2 hours at room temperature

  • Increase washing steps to 4x5 minutes with TBST to reduce background

• Detection Method:

  • For enhanced sensitivity, chemiluminescent detection is recommended

  • For quantitative analysis, consider fluorescent secondary antibodies

  • Use appropriate secondary antibody (anti-rabbit IgG) at 1:5000-1:10000 dilution

• Troubleshooting:

  • If multiple bands appear, consider potential detection of pseudogene products or different isoforms

  • If weak signal, extend exposure time or increase antibody concentration

  • If high background, increase blocking time and washing steps

A systematic approach should start with the manufacturer's recommended conditions and adjust one parameter at a time while keeping others constant.

What are best practices for immunohistochemistry protocols using the PPP1R2 (Ab-120/121) Antibody?

For optimal IHC results with the PPP1R2 (Ab-120/121) Antibody, follow these methodological guidelines:

• Tissue Preparation and Antigen Retrieval:

  • Use formalin-fixed, paraffin-embedded (FFPE) sections (4-6 μm thickness)

  • Perform heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) for 15-20 minutes

  • Allow sections to cool slowly to room temperature (approximately 20 minutes)

• Blocking and Antibody Dilution:

  • Block with 5% normal goat serum in PBS for 1 hour at room temperature

  • Start with 1:50 dilution for the primary antibody (adjust based on signal intensity)

  • Dilute antibody in blocking buffer containing 0.3% Triton X-100 for better penetration

  • Incubate sections with primary antibody overnight at 4°C in a humidified chamber

• Controls and Validation:

  • Include human breast carcinoma tissue as a positive control (verified in product data)

  • Include a negative control by omitting primary antibody

  • Consider using peptide competition controls with the immunizing peptide

• Detection System:

  • Use a polymer-based detection system for optimal signal-to-noise ratio

  • For chromogenic detection, DAB (3,3'-diaminobenzidine) is recommended

  • For fluorescent detection, Alexa Fluor conjugated secondary antibodies provide greater sensitivity

• Counterstaining and Mounting:

  • For brightfield microscopy, counterstain with hematoxylin for 1-2 minutes

  • Mount with permanent mounting medium for long-term preservation

  • For fluorescence, use DAPI nuclear counterstain and anti-fade mounting medium

Optimization is critical as different tissue types may require adjustment of dilution, incubation time, or antigen retrieval methods.

How can phosphorylation status of PPP1R2 be monitored using the Ab-120/121 antibody?

Monitoring the phosphorylation status of PPP1R2 using the Ab-120/121 antibody requires specialized approaches:

• Preparation of Phosphorylated and Non-phosphorylated Controls:

  • Generate in vitro phosphorylated PPP1R2 using purified glycogen synthase kinase 3 (GSK3)

  • Treat cell lysates with lambda phosphatase to create dephosphorylated controls

  • Use both controls to validate antibody specificity for phosphorylation state

• Phos-tag™ SDS-PAGE Approach:

  • Incorporate Phos-tag™ acrylamide into SDS-PAGE gels

  • This causes mobility shift of phosphorylated proteins

  • Run phosphorylated and non-phosphorylated controls alongside samples

  • Transfer and blot with PPP1R2 (Ab-120/121) antibody

  • Analyze band migration patterns to determine phosphorylation status

• Combined Antibody Approach:

  • Use phospho-specific antibodies targeting Ser120/121 alongside PPP1R2 (Ab-120/121)

  • Compare signal ratios to determine relative phosphorylation levels

  • Consider using PPP1R2 (Ab-120/121) for total protein and phospho-specific for modified form

• Functional Correlation:

  • Monitor PP1 activity using phosphatase assays in parallel

  • Correlate phosphorylation status with inhibitory activity

  • Remember that dephosphorylated PPP1R2 is the active inhibitory form of PP1

This multi-faceted approach provides comprehensive information about PPP1R2 phosphorylation status and its functional implications in cellular processes.

What positive and negative controls should be used with PPP1R2 (Ab-120/121) Antibody?

Implementing appropriate controls is essential for validating experimental results with the PPP1R2 (Ab-120/121) Antibody:

Positive Controls:

• Cell Line Lysates:

  • Jurkat and JK cell extracts (verified in product validation data)

  • Human breast carcinoma tissue (for IHC applications)

  • Cell lines with known high PPP1R2 expression (testis cell lines, spermatogenic cells)

• Recombinant Protein:

  • Purified recombinant PPP1R2 protein

  • In vitro phosphorylated and non-phosphorylated versions

• Tissue Samples:

  • Testis tissue sections (PPP1R2 is highly expressed in developing spermatogenic cells)

  • Human breast tissue (confirmed in product validation)

Negative Controls:

• Antibody Specificity Controls:

  • Peptide competition with the immunizing peptide (Q-E-S(p)-S(p)-G-E)

  • Secondary antibody only control (omit primary antibody)

  • Isotype control (non-specific rabbit IgG at same concentration)

• Sample-based Controls:

  • PPP1R2 knockdown or knockout samples (siRNA or CRISPR-based)

  • Tissues with minimal PPP1R2 expression (based on tissue expression databases)

• Processing Controls:

  • For IHC: tissue sections processed without antigen retrieval

  • For WB: samples without reducing agent to confirm specificity is not dependent on protein reduction state

Western blot analysis with the PPP1R2 (Ab-120/121) antibody has shown that the signal can be eliminated when the antibody is pre-incubated with the synthesized peptide, confirming specificity .

How does fixation method affect epitope recognition by PPP1R2 (Ab-120/121) Antibody in IHC?

The fixation method significantly impacts epitope recognition by PPP1R2 (Ab-120/121) Antibody in immunohistochemistry:

• Formalin Fixation (10% Neutral Buffered Formalin):

  • Recommended fixation method for most applications

  • Creates protein cross-links that may mask the epitope

  • Requires heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0)

  • Fixation time should be optimized (12-24 hours typically sufficient)

  • Excessive fixation (>48 hours) may irreversibly mask the epitope

• Paraformaldehyde Fixation (4% PFA):

  • Alternative for fresh frozen sections and cell preparations

  • Generally provides good epitope preservation

  • Shorter fixation times (10-20 minutes) for cultured cells

  • May require milder antigen retrieval methods

• Methanol/Acetone Fixation:

  • Not recommended for this antibody

  • May denature phospho-epitopes

  • Could potentially affect recognition of the Ser120/121 region

  • If used, validation against formalin-fixed samples is essential

• Fresh Frozen Sections:

  • Minimal epitope masking, potentially higher sensitivity

  • Post-fixation with 4% PFA recommended for tissue integrity

  • May exhibit higher background compared to FFPE sections

• Antigen Retrieval Optimization by Fixation Type:

  • For formalin/PFA: Heat-induced epitope retrieval with citrate buffer (pH 6.0), 95-100°C for 15-20 minutes

  • Alternative for formalin: Tris-EDTA buffer (pH 9.0) if citrate buffer yields insufficient signal

  • For frozen sections: Milder retrieval (80°C for 10 minutes) or no retrieval

Experimental validation comparing different fixation methods is recommended when establishing a new IHC protocol with this antibody.

How can the PPP1R2 (Ab-120/121) Antibody be used to investigate PP1 regulation in cancer models?

Investigating PP1 regulation in cancer models using the PPP1R2 (Ab-120/121) Antibody requires a multifaceted approach:

• Expression Profiling in Cancer Tissues:

  • Perform IHC analysis on tissue microarrays containing multiple cancer types

  • Compare PPP1R2 expression and localization between tumor and adjacent normal tissues

  • Correlate with clinical parameters (stage, grade, patient outcome)

  • Use human breast carcinoma as a validated positive control tissue

• Phosphorylation Status Analysis in Tumor Models:

  • Analyze Ser120/121 phosphorylation in cancer cell lines using Western blot

  • Compare phosphorylation levels between normal and cancer cells

  • Correlate with PP1 activity using phosphatase assays

  • Remember that dephosphorylated PPP1R2 is the active inhibitory form of PP1

• Co-immunoprecipitation Studies for Protein-Protein Interactions:

  • Use PPP1R2 (Ab-120/121) antibody to immunoprecipitate PPP1R2 complexes

  • Analyze PP1 isoform binding preferences in different cancer models

  • Identify differentially associated proteins in normal vs. cancer states

  • Validate key interactions with reciprocal co-IP experiments

• Functional Impact of PPP1R2 in Cancer:

  • Implement PPP1R2 knockdown/overexpression in cancer cell lines

  • Monitor changes in:

    • Cell proliferation and cell cycle progression

    • Colony formation ability

    • Migration and invasion capacity

    • Drug sensitivity profiles

  • Use the antibody to confirm knockdown/overexpression efficiency

• Regulation by Upstream Kinases (GSK3) in Cancer Context:

  • Assess GSK3 activity in cancer models

  • Correlate with PPP1R2 phosphorylation status

  • Test GSK3 inhibitors and monitor impact on PPP1R2 phosphorylation

  • Link to downstream PP1 activity and cancer-relevant substrates

This approach is particularly relevant as dysregulation of PPP1R2 has been associated with various diseases, including cancer and developmental disorders .

What approaches can resolve potential cross-reactivity with PPP1R2 pseudogenes when using the Ab-120/121 antibody?

Resolving potential cross-reactivity with PPP1R2 pseudogenes requires strategic experimental design:

These approaches provide multiple layers of validation to distinguish between PPP1R2 and potential pseudogene products when using the Ab-120/121 antibody.

How can PPP1R2 (Ab-120/121) Antibody be used in combination with other techniques to study protein-protein interactions?

Combining the PPP1R2 (Ab-120/121) Antibody with complementary techniques creates a powerful approach for studying protein-protein interactions:

• Co-Immunoprecipitation (Co-IP) Applications:

  • Use PPP1R2 (Ab-120/121) antibody as the capture antibody

  • Optimize antibody-to-bead ratio (typically 2-10 μg per 50 μl of protein A/G beads)

  • Include peptide competition controls to verify specificity

  • Probe for interacting partners (PP1 isoforms, GSK3, other regulatory proteins)

  • Perform reciprocal IPs to validate interactions

• Proximity Ligation Assay (PLA):

  • Combine PPP1R2 (Ab-120/121) with antibodies against potential interacting partners

  • Visualize protein interactions in situ with subcellular resolution

  • Quantify interaction signals across different cell types or experimental conditions

  • Compare interactions in normal versus disease states

  • Protocol modification: use 1:100 dilution for PPP1R2 antibody in PLA applications

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs of PPP1R2 and potential partners

  • Validate constructs using the antibody to confirm expression

  • Use the antibody in parallel immunofluorescence to correlate BiFC signals with total PPP1R2

  • Optimize fixation conditions to preserve both BiFC signal and antibody epitope

• Chromatin Immunoprecipitation (ChIP) for Nuclear Interactions:

  • Use PPP1R2 antibody for ChIP to identify potential chromatin associations

  • Perform sequential ChIP (ChIP-reChIP) to identify co-occupancy with PP1

  • Follow with mass spectrometry to identify novel nuclear interactors

  • Validate findings with GSK3 inhibition experiments to manipulate PPP1R2 phosphorylation

These integrated approaches provide complementary data on PPP1R2 interactions, offering insights into both stable and transient protein complexes across different cellular contexts.

What are methodological considerations when using PPP1R2 (Ab-120/121) Antibody in studies of testicular and sperm function?

Studies of testicular and sperm function using PPP1R2 (Ab-120/121) Antibody require specialized methodological considerations:

• Sample Preparation for Testicular Tissue:

  • Use Bouin's fixative for optimal preservation of testicular architecture

  • Limit fixation time to 12-18 hours to prevent over-fixation

  • Process tissue within 30 minutes of collection to preserve phosphorylation status

  • Consider cryosectioning for phospho-epitope preservation

  • Modified antigen retrieval: use Tris-EDTA buffer (pH 9.0) for 10 minutes

• Isolation and Analysis of Spermatogenic Cell Populations:

  • Separate testicular cells by velocity sedimentation or flow cytometry

  • Prepare nuclear and cytoplasmic fractions separately

  • Add phosphatase inhibitors to all buffers

  • Perform Western blot analysis on distinct spermatogenic cell populations

  • Use 1:750 dilution of antibody for these specific applications

• Sperm Protein Extraction Optimization:

  • Use specialized extraction buffers containing 6M urea for sperm proteins

  • Include 1% Triton X-100 and 0.5% SDS in extraction buffer

  • Sonicate briefly (3x10s) to aid protein extraction

  • Add reducing agents (5mM DTT) to extraction buffer

  • For proteomic studies, avoid heat-treatment of samples

• Differentiating Between PPP1R2 and Testis-Specific Isoforms/Pseudogenes:

  • Design validation experiments with tissues expressing PPP1R2 but not pseudogenes

  • Use RT-PCR to correlate protein detection with transcript expression

  • Compare antibody reactivity with known expression patterns of PPP1R2P3 and PPP1R2P9 in sperm

  • Consider complementary approaches using antibodies to other regions

• Co-localization Studies in Testis and Sperm:

  • Combine with antibodies against PP1γ2 (testis-specific PP1 isoform)

  • Use confocal microscopy with 0.3-0.5μm optical sections

  • Implement super-resolution microscopy for detailed localization

  • Quantify co-localization using Pearson's coefficient analysis

  • Control for autofluorescence (common in testicular tissue)

These specialized considerations address the unique challenges of studying PPP1R2 in reproductive tissues and provide methodological guidance for obtaining reliable results in this specific research context.

How can quantitative analyses be performed using PPP1R2 (Ab-120/121) Antibody in immunofluorescence studies?

Performing quantitative analyses with PPP1R2 (Ab-120/121) Antibody in immunofluorescence requires standardized approaches:

• Standardization of Immunofluorescence Protocol:

  • Use consistent fixation method (4% paraformaldehyde, 15 minutes)

  • Standardize antibody concentration (1:75 dilution recommended for IF)

  • Process all samples in parallel using identical reagents

  • Include calibration standards in each experiment

  • Image all samples with identical microscope settings

• Image Acquisition Parameters:

  • Use confocal microscopy with defined settings (pinhole, gain, offset)

  • Capture Z-stacks (0.5μm steps) to ensure complete signal representation

  • Avoid pixel saturation (keep intensity values below 95% of dynamic range)

  • Include flat-field correction to account for illumination non-uniformities

  • Capture multiple random fields (minimum 10) per sample for statistical validity

• Signal Quantification Methods:

  • Mean Fluorescence Intensity (MFI):

    • Define regions of interest (ROIs) based on cellular compartments

    • Measure average intensity within ROIs

    • Subtract local background from each measurement

    • Calculate nuclear:cytoplasmic ratio of PPP1R2 signal

  • Integrated Density Measurement:

    • Multiply mean intensity by area for total signal quantification

    • Particularly useful for heterogeneously distributed signals

    • Normalize to cell number or nuclear count

  • Co-localization Quantification:

    • Calculate Pearson's correlation coefficient or Mander's overlap coefficient

    • Use object-based co-localization for punctate structures

    • Report percentage of PPP1R2-positive structures co-localized with partners

• Analysis Software and Methods:

  • Use specialized software (ImageJ/Fiji with Bio-Formats plugin)

  • Create standardized macros for batch processing

  • Implement automated thresholding methods (Otsu, Li, etc.)

  • Apply watershed segmentation for touching objects

  • Validate automated analysis against manual measurements

• Controls for Quantitative Analysis:

  • Include signal calibration standards in each experiment

  • Process siRNA knockdown samples for signal specificity validation

  • Use phosphatase-treated samples as controls for phospho-specificity

  • Analyze technical replicates to assess measurement precision

This systematic approach ensures reliable quantitative data from immunofluorescence studies using PPP1R2 (Ab-120/121) Antibody across different experimental conditions and cell types.