PPP1CB Antibody

What is PPP1CB and what cellular functions does it regulate?

PPP1CB, also known as PP-1B, belongs to the PPP phosphatase family and PP-1 subfamily. It functions as a catalytic subunit of protein phosphatase 1 (PP1), which is essential for several cellular processes. PPP1CB participates in the regulation of glycogen metabolism, muscle contractility, and protein synthesis . It is particularly important in cell division processes and is involved in the regulation of ionic conductivity . Recent research has also implicated PPP1CB in cardiac function, where it plays a unique role in affecting cardiac contractile function through regulation of myofilament protein phosphorylation . Unlike other PP1 isoforms, PPP1CB shows enriched localization to sarcomeres, suggesting specialized functional roles in muscle tissues .

What are the validated applications for PPP1CB antibodies?

PPP1CB antibodies have been validated for multiple experimental applications, with different products showing specific performance characteristics. The 55136-1-AP antibody, for example, has been validated for:

Additionally, specialized antibodies like 84938-1-PBS are designed as part of matched antibody pairs for cytometric bead array applications . For optimal results in any application, antibody dilutions should be titrated for each specific experimental system .

How should PPP1CB antibodies be stored to maintain reactivity?

Storage conditions are critical for maintaining antibody reactivity and integrity. For most PPP1CB antibodies, including the 55136-1-AP product, the recommended storage conditions are:

• Store at -20°C

• Stable for one year after shipment

• Aliquoting is generally unnecessary for -20°C storage

• Some products contain 0.1% BSA as a stabilizer

For specialized formulations like the 84938-1-PBS antibody (PBS only, BSA and azide free format), more stringent storage is required:

• Store at -80°C

• Maintain in PBS-only formulation until ready for conjugation

Proper storage ensures long-term stability and consistent experimental results across multiple studies.

How can PPP1CB knockdown experiments be designed and validated?

Designing effective PPP1CB knockdown experiments requires careful consideration of cell types, knockdown methods, and validation approaches. Based on published research:

• siRNA Transfection Protocol:

  • Seed cells at 3 × 10^5 cells/well in 6-well plates

  • Transfect with si-PPP1CB or si-NC (negative control)

  • Confirm knockdown efficiency through Western blot analysis

• Protein Extraction and Validation:

  • Extract proteins using cell lysis buffer (e.g., Cell Signaling Technology)

  • Determine protein concentration using bicinchoninic acid protein assay kit

  • Separate 40 μg protein/lane by 10% SDS-PAGE

  • Transfer to PVDF membranes

  • Block with TBST containing 5% bovine serum albumin

  • Incubate with primary antibody against PPP1CB

  • Perform standard Western blot detection procedures

• Functional Validation:

  • Perform wound healing assays to assess effects on cell migration

  • Create a wound in cell monolayer using a micropipette tip

  • Wash with PBS and photograph at 0 hours and 48 hours

  • Calculate and quantify the ratio of residual wound area to initial area

These protocols have successfully demonstrated that PPP1CB knockdown significantly reduces migration and invasion of pancreatic adenocarcinoma cells, validating its role in cancer progression .

What are the implications of PPP1CB expression in cancer research?

PPP1CB has emerging significance in cancer research, particularly in pancreatic adenocarcinoma (PAAD). Evidence indicates that:

Understanding these patterns may help identify new therapeutic targets and prognostic indicators in pancreatic cancer research.

How do PPP1CB knockout models differ from knockdown approaches in cardiac research?

Cardiac research on PPP1CB utilizes both knockdown and knockout approaches, each with distinct advantages and findings:

• Knockout Models:

  • Cardiac-specific deletion using either Nkx2.5-Cre (embryonic) or tamoxifen-inducible αMHC-MerCreMer (adult-specific)

  • Complete deletion of the PPP1CB gene rather than temporary reduction

  • Results in concentric cardiac remodeling, interstitial fibrosis, and contractile dysregulation

  • Interestingly, isolated myocytes from PPP1CB-deleted hearts show enhanced contractility

• Mechanistic Findings from Knockout Models:

  • No effect on phospholamban phosphorylation

  • No alteration in Ca2+ handling dynamics in adult myocytes

  • Elevated phosphorylation of myofilament proteins including myosin light chain 2 and cardiac myosin binding protein C

  • Suggests a unique role for PPP1CB in regulating myofilament function

• Isoform Specificity:

  • Deletion of PPP1CA (PP1α) or PPP1CC (PP1γ) has minimal effect on the whole heart

  • Only PPP1CB deletion results in significant cardiac phenotypes

  • Demonstrates non-redundant functions of PP1 isoforms in cardiac tissue

These findings highlight the importance of considering both temporary (knockdown) and permanent (knockout) approaches when studying phosphatase function in complex tissues.

How should researchers select between polyclonal and recombinant monoclonal antibodies for PPP1CB detection?

The choice between polyclonal and recombinant monoclonal antibodies for PPP1CB detection depends on experimental goals and required specificity:

When selecting an antibody:

• Consider experimental application:

  • For standard detection in common techniques like Western blot, polyclonal antibodies often provide sufficient sensitivity

  • For quantitative applications requiring precise epitope targeting, recombinant monoclonal antibodies offer superior consistency

• Evaluate species reactivity:

  • Polyclonal 55136-1-AP shows reactivity with human, mouse, and rat samples

  • Recombinant monoclonal 84938-1-PBS is validated specifically for human targets

• Assess technical requirements:

  • For conjugation-dependent applications, specialized formats like 84938-1-PBS (PBS only, BSA and azide free) are preferable

  • For routine detection without conjugation needs, standard formulations are suitable

What are the optimal protocols for immunohistochemical detection of PPP1CB in tissue samples?

For optimal immunohistochemical detection of PPP1CB in tissue samples, researchers should follow these validated protocols:

• Antigen Retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Complete retrieval before antibody incubation

• Antibody Dilution:

  • For polyclonal antibody 55136-1-AP: Use 1:400-1:1600 dilution

  • Titrate for each tissue type to optimize signal-to-noise ratio

• Scoring System for Quantification:

  • Implement standardized intensity scoring:

    • 0 points (negative)

    • 1 point (+)

    • 2 points (++)

    • 3 points (+++)

  • Calculate total score as product of staining intensity and staining rate

  • Categorize samples with score <1.2 as low expression

  • Categorize samples with score ≥1.2 as high expression

• Documentation and Analysis:

  • Use an Aperio scanner or equivalent imaging system (magnification, x200)

  • Have two professional pathologists score independently to ensure reliability

  • Evaluate both cytoplasmic and nuclear staining independently

These protocols have been validated in studies examining PPP1CB expression in pancreatic adenocarcinoma and can be adapted for other tissue types.

How can researchers troubleshoot unexpected molecular weight observations for PPP1CB in Western blots?

When troubleshooting unexpected molecular weight observations for PPP1CB in Western blots, researchers should consider:

• Expected vs. Observed Weights:

  • Calculated molecular weight: 37 kDa

  • Observed molecular weight range: 33-40 kDa

  • Variation may reflect post-translational modifications or isoform expression

• Sample Preparation Considerations:

  • Ensure complete protein denaturation with appropriate SDS and heating

  • Use fresh protease inhibitors in lysis buffer

  • Consider phosphatase inhibitors to preserve phosphorylation state

  • Run proper molecular weight markers alongside samples

• Technical Solutions for Common Issues:

  • Multiple bands: May represent isoforms, degradation products, or post-translational modifications

  • No signal: Verify transfer efficiency with reversible staining

  • High background: Optimize blocking conditions and antibody dilutions

  • Unexpected size: Confirm antibody specificity with positive controls

• Validation Approaches:

  • Compare results across multiple cell lines with known PPP1CB expression

  • Include validated positive controls (e.g., A549 cells, BxPC-3 cells)

  • Consider knockdown/knockout validation if available

Consistent with product documentation, the observed molecular weight of PPP1CB may vary slightly from the calculated weight due to biological factors like post-translational modifications.

How is PPP1CB antibody used in protein-protein interaction studies?

PPP1CB antibody plays a critical role in protein-protein interaction studies through several methodological approaches:

These approaches provide comprehensive insights into PPP1CB function within complex cellular networks and disease states.

What role does PPP1CB play in myofilament phosphorylation and cardiac function?

PPP1CB plays a specialized role in regulating myofilament phosphorylation and cardiac function, distinct from other PP1 isoforms:

• Isoform-Specific Cardiac Effects:

  • Deletion of PPP1CB results in concentric remodeling of the heart, interstitial fibrosis, and contractile dysregulation

  • In contrast, deletion of PPP1CA (PP1α) or PPP1CC (PP1γ) has minimal effect on the whole heart

  • This demonstrates a unique non-redundant function for PPP1CB in cardiac tissue

• Subcellular Localization and Targets:

  • PPP1CB shows enriched localization to sarcomeres compared to other PP1 isoforms

  • Loss of PPP1CB results in elevated phosphorylation of myofilament proteins including:

    • Myosin light chain 2

    • Cardiac myosin binding protein C

• Contractility Regulation:

  • Myocytes isolated from PPP1CB-deleted hearts show enhanced contractility

  • This effect occurs without changes in phospholamban phosphorylation

  • Ca²⁺ handling dynamics remain unaltered in adult myocytes from PPP1CB-deleted hearts

• Mechanistic Model:

  • PPP1CB likely regulates myofilament protein phosphorylation directly

  • Changes in phosphorylation state secondarily influence cardiac contractility or relaxation

  • This represents a distinct regulatory pathway from the classical phospholamban-mediated mechanisms

These findings highlight PPP1CB as a potential therapeutic target for modulating cardiac contractility in heart disease states.

How might PPP1CB antibodies be incorporated into multiplex assays for clinical biomarker discovery?

PPP1CB antibodies are increasingly being incorporated into multiplex assays for clinical biomarker discovery through several innovative approaches:

• Matched Antibody Pair Technology:

  • Specialized products like 84938-1-PBS are designed specifically as part of matched antibody pairs

  • For example, MP01723-1 utilizes 84938-1-PBS as capture antibody and 84938-2-PBS as detection antibody

  • These have been validated in cytometric bead array applications

• Conjugation-Ready Formulations:

  • Recombinant antibodies in PBS-only (BSA and azide-free) storage buffer are prepared at a concentration of 1 mg/mL

  • This format makes them ready for conjugation to various detection systems

  • Enables application in ELISAs, multiplex assays, mass cytometry, and multiplex imaging applications

• Integration with Clinical Data:

  • PPP1CB expression analysis can be correlated with clinical outcomes

  • This approach was successfully used in PAAD research to evaluate PPP1CB as a prognostic indicator

  • Statistical methods included Kaplan‐Meier survival curves and univariate survival analysis using log-rank tests

• Standardization Requirements:

  • Recombinant production enables unrivaled batch-to-batch consistency

  • This consistency is essential for long-term biomarker studies

  • Facilitates easy scale-up and future security of supply

The development of these technologies positions PPP1CB antibodies as valuable tools in clinical research and potential diagnostic applications.

What emerging techniques are enhancing the specificity of phosphatase targeting in cell signaling research?

Several emerging techniques are enhancing the specificity of phosphatase targeting in cell signaling research, with relevance to PPP1CB studies:

• CRISPR-Cas9 Gene Editing:

  • Enables precise deletion of specific PP1 isoforms

  • Permits development of conditional knockout models with temporal control

  • Research using Nkx2.5-Cre and αMHC-MerCreMer alleles demonstrates the power of tissue-specific and temporally controlled gene deletion

• Isoform-Specific Inhibition Strategies:

  • Development of small molecules targeting specific PP1 isoforms

  • Peptide inhibitors designed to disrupt specific PP1-regulatory subunit interactions

  • These approaches offer greater specificity than traditional phosphatase inhibitors

• Advanced Imaging Techniques:

  • Fluorescent biosensors to monitor phosphatase activity in real-time

  • Super-resolution microscopy to visualize subcellular localization of PPP1CB

  • These techniques can reveal the spatial and temporal dynamics of PPP1CB activity

• Quantitative Phosphoproteomics:

  • Mass spectrometry-based identification of PPP1CB substrates

  • Comparison of phosphopeptide abundance in wild-type versus PPP1CB-deficient samples

  • This approach can identify novel substrates and signaling pathways regulated by PPP1CB

These advanced techniques are expanding our understanding of PPP1CB's role in complex signaling networks and providing new opportunities for therapeutic intervention.