PPP1CC Antibody

Fundamental Research Questions About PPP1CC Antibodies

What is PPP1CC and why is it important in research?

PPP1CC is a catalytic subunit of protein phosphatase 1 (PP1), belonging to the PPP phosphatase family. It's essential for cell division and participates in regulating glycogen metabolism, muscle contractility, and protein synthesis . The significance of PPP1CC in research stems from its ubiquitous role in dephosphorylating hundreds of biological targets through association with over 200 regulatory proteins to form highly specific holoenzymes . PPP1CC also plays critical roles in:

• Regulation of ionic conductances and long-term synaptic plasticity

• Dephosphorylation of substrates like postsynaptic density-associated Ca²⁺/calmodulin-dependent protein kinase II

• Chromatin structure control and cell cycle progression during the transition from mitosis to interphase as part of the PTW/PP1 phosphatase complex

The Ppp1cc gene's isoforms have distinct expression patterns and functions, making them important targets for understanding tissue-specific phosphatase regulation .

What are the main isoforms of PPP1CC and how do they differ?

The Ppp1cc gene encodes two alternatively spliced variants:

These isoforms show non-overlapping expression patterns in testis, with PPP1CC2 being the only PP1 isoform not detected in Sertoli cells and spermatogonia, while PPP1CC1 is absent in postmeiotic germ cells . This distinct expression pattern explains why global deletion of Ppp1cc primarily affects male fertility, as other PP1 isoforms cannot compensate for the loss of PPP1CC2 in postmeiotic germ cells .

How do I choose the right PPP1CC antibody for my research?

Selecting the appropriate PPP1CC antibody requires consideration of several factors:

• Target specificity: Determine whether you need an antibody that recognizes both PPP1CC isoforms or one that is isoform-specific

• Application compatibility: Verify the antibody has been validated for your intended application (WB, IP, IHC, IF/ICC)

• Species reactivity: Ensure the antibody recognizes PPP1CC from your species of interest

• Clonality: Consider whether polyclonal (broader epitope recognition) or monoclonal (higher specificity) is more appropriate for your experiments

• Validated method data: Review validation data for your specific application method

For example, if studying PPP1CC in mouse brain tissue via Western blot, Proteintech's 11082-1-AP antibody has been validated for this application and species at a recommended dilution of 1:2000-1:16000 .

Experimental Design and Methodology

What are the optimal conditions for Western blotting with PPP1CC antibodies?

For optimal Western blotting results with PPP1CC antibodies:

For example, in a study examining circular RNA PPP1CC, researchers extracted total proteins using RIPA buffer, measured concentrations using bicinchoninic acid method, separated proteins on 10% SDS-PAGE, and transferred to PVDF membranes before blocking with 5% skim milk for 1.5 hours .

How can I optimize immunohistochemistry protocol for PPP1CC detection?

For optimal immunohistochemistry (IHC) with PPP1CC antibodies:

When performing IHC for PPP1CC localization studies, researchers have identified distinct expression patterns, with PPP1CC1 localized in Sertoli cells appearing as radial spoke-like structures radiating from the periphery toward the lumen of the seminiferous epithelium, while PPP1CC2 is expressed in pachytene spermatocytes and postmeiotic developing germ cells .

What controls should I include when validating a new PPP1CC antibody?

When validating a new PPP1CC antibody, include the following controls:

• Positive tissue/cell controls:

  • Brain tissue (mouse/rat) - consistently shows strong PPP1CC expression

  • HEK-293 or HeLa cells - verified positive for many PPP1CC antibodies

• Negative controls:

  • Primary antibody omission - to assess non-specific binding of secondary antibody

  • Isotype control - matching the primary antibody's host species and isotype

• Knockout/knockdown validation:

  • Ppp1cc knockout/knockdown samples when available - provides strongest specificity validation

  • Example: Using Ppp1cc knockout mice to confirm antibody specificity

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Should eliminate or significantly reduce specific signal

• Molecular weight verification:

  • PPP1CC should be detected at approximately 35-37 kDa

  • Multiple bands may indicate isoforms or degradation products

Knockout validation is particularly valuable, as demonstrated in a study where Invitrogen PPP1CC antibody (PA5-21671) was validated using mouse knockout samples .

Advanced Research Applications and Considerations

How can I distinguish between PPP1CC isoforms in my experiments?

Distinguishing between PPP1CC isoforms requires specific strategies:

• Isoform-specific antibodies:

  • Use antibodies raised against unique C-terminal regions that differ between PPP1CC1 and PPP1CC2

  • Example: Abcam's sheep polyclonal antibody (ab16387) targets the C-terminus of PPP1CC

• Expression pattern analysis:

  • PPP1CC1 is expressed in Sertoli cells and premeiotic germ cells

  • PPP1CC2 is exclusively expressed in meiotic and postmeiotic germ cells

  • In immunohistochemistry of testis sections, look for characteristic staining patterns:

    • PPP1CC1: Staining in radial spoke-like structures of Sertoli cells and along the periphery of seminiferous tubules

    • PPP1CC2: Staining in pachytene spermatocytes and subsequent stages of postmeiotic germ cells

• Tissue-specific expression:

  • Testis-specific sample fractionation can help distinguish isoforms

  • Western blot analysis of extracts from isolated Sertoli cells will show PPP1CC1 but not PPP1CC2

  • Extracts from spermatozoa will show PPP1CC2 but not PPP1CC1

• RT-PCR approach:

  • Design primers specific to unique regions of each isoform

  • Example primers for detecting circular RNA of PPP1CC: forward 5′-CAGGAGAACTGTTGATGGCATA-3′, reverse 5′-ATACCC CTTGGAGGCGTTAC-3′

What are the key considerations when studying PPP1CC in fertility research?

When investigating PPP1CC in fertility research, consider:

• Isoform-specific expression patterns:

  • PPP1CC2 is the predominant testicular isoform in postmeiotic cells

  • PPP1CC1 is present in Sertoli cells and premeiotic germ cells

  • Other PP1 isoforms (PPP1CA, PPP1CB) are expressed in testis but cannot compensate for loss of PPP1CC2

• Knockout models interpretation:

  • Global Ppp1cc null (-/-) mice are infertile due to impaired spermatogenesis

  • Conditional knockout of Ppp1cc in germ cells (using Stra8-Cre) results in oligo-terato-asthenozoospermia and male infertility

  • Phenotype comparison between global and conditional knockouts helps distinguish somatic vs. germ cell-specific effects

• Cell-specific analysis:

  • Western blot analysis of Sertoli cells shows presence of PPP1CC1, PPP1CA, and PPP1CB, but not PPP1CC2

  • Primary cultures of spermatogonial stem cells (SSCs) express all PP1 isoforms except PPP1CC2

• Compensation mechanisms:

  • In tissues other than testis, loss of PP1 isoforms can be compensated by other isoforms

  • The exclusion of three of the four isoforms in postmeiotic testicular cells explains why PPP1CC2 is the only PP1 isoform found in spermatozoa

How can I troubleshoot non-specific binding with PPP1CC antibodies?

When encountering non-specific binding with PPP1CC antibodies:

• Optimize antibody dilution:

  • Test a dilution series based on manufacturer recommendations

  • Example ranges: 1:500-1:16000 for WB , 1:20-1:500 for IHC

• Modify blocking conditions:

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (from 1 hour to overnight)

  • Example: 5% skim milk powder for 1.5 hours has been reported effective

• Adjust washing stringency:

  • Increase washing duration or number of washes

  • Add low concentrations of detergent (0.1-0.3% Tween-20) to wash buffer

• Sample preparation improvements:

  • Ensure complete protein denaturation for WB

  • Optimize fixation protocols for IHC/IF

  • Use fresh samples to avoid degradation products

• Validation approaches:

  • Peptide competition assay to identify non-specific binding

  • Use knockout or knockdown samples as negative controls

  • Test alternative antibodies targeting different epitopes

• Cross-reactivity assessment:

  • Check sequence homology between PPP1CC and other PP1 isoforms

  • PPP1CA and PPP1CB have high sequence similarity to PPP1CC

  • Consider using antibodies targeting unique regions like the C-terminus

Data Interpretation and Experimental Design Considerations

How do I interpret changes in PPP1CC levels across different experimental conditions?

When interpreting changes in PPP1CC levels:

• Consider isoform-specific changes:

  • PPP1CC1 and PPP1CC2 may be differentially regulated

  • Verify which isoform your antibody detects (or if it detects both)

  • Example: In Ppp1cc knockout studies, PPP1CA expression was upregulated in testis, suggesting compensatory mechanisms

• Tissue/cell type context:

  • Expression patterns are tissue-specific (e.g., PPP1CC2 is predominantly in testis)

  • Cellular localization may change with experimental conditions

  • Example: PPP1CC1 shows specific localization in Sertoli cells as radial spoke-like structures

• Normalization considerations:

  • Use appropriate loading controls (GAPDH, actin, tubulin)

  • Consider normalizing to total protein using stain-free gels or membrane staining

  • Verify that loading controls aren't affected by your experimental conditions

• Statistical analysis:

  • Perform quantification across multiple biological replicates

  • Example: Studies examining circular RNA PPP1CC performed experiments in triplicate and analyzed data using two-way ANOVA with Tukey test confirmation

• Functional correlation:

  • Correlate changes in PPP1CC levels with substrate phosphorylation status

  • Link changes to downstream functional effects (e.g., cell proliferation, meiotic progression)

What are the best approaches for studying PPP1CC-protein interactions?

For studying PPP1CC-protein interactions:

• Immunoprecipitation (IP):

  • Several antibodies have been validated for IP of PPP1CC:

    • Proteintech 11082-1-AP: Use 0.5-4.0 μg for 1.0-3.0 mg total protein lysate

    • Proteintech 55150-1-AP: Validated for IP from mouse brain tissue

  • After IP, analyze by Western blot or mass spectrometry to identify interactors

• Proximity ligation assay (PLA):

  • Detects protein-protein interactions in situ with high sensitivity

  • Requires antibodies from different host species against PPP1CC and its potential interactor

  • Produces fluorescent spots where proteins interact (<40nm proximity)

• Co-immunofluorescence:

  • Use validated antibodies for immunofluorescence:

    • Proteintech 55150-1-AP: 1:50-1:500 dilution for IF/ICC in HeLa cells

    • Proteintech 11082-1-AP: 1:50-1:500 dilution for IF/ICC in HEK-293 cells

  • Analyze colocalization using confocal microscopy

• Pull-down assays:

  • Use recombinant PPP1CC as bait to identify interacting proteins

  • Can be performed with tagged PPP1CC constructs (His, GST, etc.)

• Yeast two-hybrid screening:

  • Can identify novel PPP1CC interactors from cDNA libraries

  • Validate interactions using methods above in mammalian cells

How can I design experiments to study PPP1CC's role in signaling pathways?

When designing experiments to study PPP1CC's role in signaling:

• Pathway-specific activation/inhibition:

  • Use pathway stimulators/inhibitors to trigger phosphorylation events

  • Monitor PPP1CC recruitment to signaling complexes using IP or IF

  • Example pathways: insulin signaling, MAPK cascades, focal adhesion

• Phosphatase activity assays:

  • Use immunoprecipitated PPP1CC to measure phosphatase activity

  • Employ phospho-specific antibodies to monitor substrate dephosphorylation

  • Example substrate: RPS6KB1 (documented target of PPP1CC)

• Knockout/knockdown approaches:

  • Use siRNA, shRNA, or CRISPR-Cas9 to deplete PPP1CC

  • Monitor impacts on pathway activity using phospho-specific antibodies

  • Example: Several PPP1CC antibodies have been validated in knockout/knockdown studies

• Isoform-specific analysis:

  • Design isoform-specific knockdowns

  • Use tissue/cell-specific conditional knockout models

  • Example: Conditional knockout of Ppp1cc in germ cells using Stra8-Cre transgenic mice

• Cellular localization during signaling:

  • Track PPP1CC localization changes upon pathway activation

  • Use subcellular fractionation or live-cell imaging

  • Example: PPP1CC participates in PTW/PP1 phosphatase complex during mitosis-to-interphase transition

Current Research Frontiers and New Methodologies

What are the emerging roles of PPP1CC beyond its classical functions?

Emerging research reveals expanded roles for PPP1CC:

• Circular RNA regulation:

  • Circular RNA PPP1CC has been implicated in Porphyromonas gingivalis-induced inflammatory responses

  • Research has identified miR-103a-3p and miR-107 as potential interactors with circular PPP1CC

• Immune system regulation:

  • PPP1CC dephosphorylates the 'Ser-418' residue of FOXP3 in regulatory T-cells (Treg) from patients with rheumatoid arthritis

  • This dephosphorylation inactivates FOXP3 and renders Treg cells functionally defective

• Circadian rhythm control:

  • PPP1CC, in balance with CSNK1D and CSNK1E, determines circadian period length

  • It regulates the speed and rhythmicity of PER1 and PER2 phosphorylation

  • May dephosphorylate CSNK1D and CSNK1E directly

• Chromatin regulation:

  • PPP1CC is a component of the PTW/PP1 phosphatase complex

  • This complex plays a role in chromatin structure control and cell cycle progression during the transition from mitosis to interphase

• Neuronal plasticity:

  • Involved in regulation of ionic conductances and long-term synaptic plasticity

  • May dephosphorylate postsynaptic density-associated Ca²⁺/calmodulin-dependent protein kinase II

How can I apply advanced imaging techniques to study PPP1CC localization and dynamics?

Advanced imaging approaches for PPP1CC studies include:

• Super-resolution microscopy:

  • STORM, PALM, or STED microscopy can resolve PPP1CC localization beyond diffraction limit

  • Particularly useful for studying PPP1CC in complex structures like the postsynaptic density or chromosomes

  • Requires highly specific antibodies validated for immunofluorescence, such as:

    • Proteintech 55150-1-AP (1:50-1:500 dilution for IF/ICC)

    • OriGene TA308937 (1:100-1:1000 for ICC/IF)

• Live-cell imaging with fluorescent fusion proteins:

  • Create PPP1CC-FP (fluorescent protein) fusions to monitor dynamics in living cells

  • Consider isoform-specific tagging to distinguish PPP1CC1 vs PPP1CC2

  • Use photoactivatable or photoconvertible tags for pulse-chase experiments

• FRET/FLIM for interaction studies:

  • Förster resonance energy transfer (FRET) or fluorescence lifetime imaging (FLIM)

  • Can detect PPP1CC interactions with regulatory subunits or substrates in real-time

  • Requires fluorophore-tagged proteins with appropriate spectral overlap

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence microscopy of PPP1CC with electron microscopy

  • Provides ultrastructural context for PPP1CC localization

  • Particularly valuable for studying PPP1CC at specialized cellular structures

• Expansion microscopy:

  • Physical expansion of fixed specimens can improve resolution with standard microscopes

  • Useful for studying PPP1CC in densely packed structures like synapses or centrosomes

  • Compatible with standard immunofluorescence protocols using validated antibodies

What are the most recent methodological advances in studying phosphatase-substrate relationships relevant to PPP1CC?

Recent methodological advances for studying PPP1CC-substrate relationships include:

• Phosphoproteomic approaches:

  • Quantitative phosphoproteomics before/after PPP1CC manipulation

  • Example workflow: SILAC labeling → PPP1CC knockdown/inhibition → phosphopeptide enrichment → LC-MS/MS

  • Enables unbiased identification of potential PPP1CC substrates

• BioID or TurboID proximity labeling:

  • Fuse PPP1CC to biotin ligase (BioID2/TurboID)

  • Allows identification of proteins in close proximity to PPP1CC in living cells

  • Can capture transient enzyme-substrate interactions difficult to detect by IP

• Engineered PPP1CC variants:

  • Create substrate-trapping mutants (phosphatase-dead)

  • Develop analog-sensitive PPP1CC for chemical genetic approaches

  • Design PPP1CC with fast- or slow-cleaving tags for rapid induction/degradation

• CRISPR-based technologies:

  • CRISPR activation/inhibition to modulate PPP1CC expression

  • CRISPR base or prime editing for generating specific PPP1CC mutations

  • CRISPR screens to identify genes affecting PPP1CC function

• Optogenetic and chemogenetic tools:

  • Create light- or drug-inducible PPP1CC recruitment systems

  • Allows temporal and spatial control of PPP1CC activity

  • Enables study of acute versus chronic effects of PPP1CC-mediated dephosphorylation