PPP1R16A Antibody

What is PPP1R16A and what is its primary function in cellular processes?

PPP1R16A (Protein Phosphatase 1 Regulatory Subunit 16A), also known as MYPT3 (Myosin Phosphatase-Targeting Subunit 3), is a regulatory protein that inhibits protein phosphatase 1 (PP1) activity toward substrates such as phosphorylase, myosin light chain, and myosin . This protein plays a critical role in controlling phosphorylation states within cells, particularly in pathways related to cell cycle regulation, apoptosis, and DNA repair . PPP1R16A is encoded by a gene located on chromosome 8 in humans and is implicated in the regulation of cell growth and survival. Its involvement in fundamental cellular processes underscores its importance as a potential therapeutic target for conditions including cancer and neurodegenerative diseases .

What types of PPP1R16A antibodies are available for research purposes?

Several types of PPP1R16A antibodies are available for research applications:

The most common types are rabbit and mouse polyclonal antibodies, with varying reactivity profiles including human, mouse, and rat samples . These antibodies are primarily available in non-conjugated formats and have been validated for applications such as Western blotting (WB), ELISA, and in some cases, immunohistochemistry (IHC) .

What are the standard applications for PPP1R16A antibodies?

The standard applications for PPP1R16A antibodies include:

• Western Blotting (WB): The most widely validated application across multiple antibody products. PPP1R16A typically appears as a band around 58 kDa in Western blots .

• Enzyme-Linked Immunosorbent Assay (ELISA): Many PPP1R16A antibodies are validated for ELISA applications with recommended dilutions typically ranging from 1:2000-1:10000 .

• Immunohistochemistry (IHC): Some antibodies are validated for detecting PPP1R16A in tissue sections, though this is less common than WB and ELISA applications .

These applications allow researchers to detect, quantify, and localize PPP1R16A in various experimental systems, providing insights into its expression patterns and potential functional roles in different cellular contexts .

How should I design validation experiments for a new PPP1R16A antibody?

A comprehensive validation strategy for PPP1R16A antibodies should include multiple approaches:

• Specificity Testing:

  • Compare signal in PPP1R16A-transfected versus non-transfected cell lysates. For example, Western blotting shows specific bands at 58 kDa in PPP1R16A-transfected 293T cells that are absent or significantly reduced in non-transfected controls .

  • Include positive control tissues known to express PPP1R16A (such as pancreas) .

  • Consider knockout/knockdown controls if available.

• Cross-Reactivity Assessment:

  • Test the antibody against closely related proteins, particularly other PP1 regulatory subunits.

  • For antibodies claiming multi-species reactivity, validate each species separately rather than assuming cross-reactivity.

• Application-Specific Validation:

  • For WB: Optimize antibody concentration (typically starting with 1:500-1:3000 dilutions) .

  • For ELISA: Determine optimal coating concentration and detection antibody dilution (typically 1:2000-1:10000) .

  • For IHC: Validate different antigen retrieval methods and antibody concentrations on known positive tissues.

• Epitope Analysis:

  • Confirm which region of PPP1R16A the antibody targets (N-terminal, C-terminal, or internal domains).

  • Consider how post-translational modifications might affect antibody binding.

Document all validation steps meticulously, including positive and negative controls, to ensure reproducibility and reliability in subsequent experiments .

What are the optimal sample preparation methods for detecting PPP1R16A in different tissue types?

Optimal sample preparation for PPP1R16A detection varies by tissue type and application:

• Cell Lysates and Tissue Homogenates for Western Blotting:

  • Use RIPA buffer supplemented with protease inhibitors for general extraction.

  • For membrane-associated fractions of PPP1R16A, consider detergent-based extraction methods.

  • Sonication or homogenization should be performed on ice to prevent protein degradation.

  • Centrifuge at 12,000-14,000g for 10-15 minutes at 4°C to remove debris.

  • Protein concentration should be determined using Bradford or BCA assays.

  • Load 25-50 μg of total protein per lane, as demonstrated in validated Western blots .

• Tissue Sections for Immunohistochemistry:

  • Formalin-fixed paraffin-embedded (FFPE) sections typically require antigen retrieval.

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes.

  • For frozen sections, fixation with 4% paraformaldehyde for 10 minutes at room temperature.

  • Block with 5-10% normal serum from the same species as the secondary antibody.

• Cell Lines for Immunocytochemistry:

  • Fix cells using 4% paraformaldehyde for 15 minutes at room temperature.

  • Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes for intracellular antigens.

  • Block with 5% BSA or normal serum for 1 hour.

Different tissues may require tissue-specific optimization. For example, pancreatic tissue lysates have been successfully used to detect PPP1R16A in Western blotting applications, while heart tissue has also shown detectable expression levels of the protein .

What are the critical parameters for achieving reproducible Western blot results with PPP1R16A antibodies?

Achieving reproducible Western blot results with PPP1R16A antibodies requires attention to several critical parameters:

• Sample Preparation:

  • Consistent protein extraction method and buffer composition.

  • Equal protein loading (25-50 μg per lane) verified by total protein staining or housekeeping proteins.

  • Fresh preparation of samples or proper storage (-80°C with minimal freeze-thaw cycles).

• Antibody Selection and Dilution:

  • Select validated antibodies with published Western blot results showing expected band size (~58 kDa) .

  • Use consistent antibody dilutions, typically in the range of 1:500-1:3000 for PPP1R16A primary antibodies .

  • Include positive controls such as PPP1R16A-transfected cell lysates when possible .

• Gel and Transfer Conditions:

  • For PPP1R16A (~58 kDa), 10% or 12% SDS-PAGE gels are appropriate.

  • Transfer using wet transfer systems at 100V for 60-90 minutes or 30V overnight at 4°C.

  • Verify transfer efficiency with reversible staining before blocking.

• Detection Method Optimization:

  • ECL detection systems have been successfully used for PPP1R16A visualization .

  • Exposure time should be optimized to avoid saturation.

  • Digital imaging systems should be calibrated for consistent sensitivity.

• Data Analysis:

  • Use densitometry software with background subtraction.

  • Normalize to appropriate loading controls.

  • Report relative values rather than absolute intensities.

Researchers have reported successful detection of PPP1R16A in human pancreas lysates and PPP1R16A-transfected 293T cell lysates using these parameters, with clear bands visible at the expected 58 kDa size .

How can I address non-specific binding when using PPP1R16A antibodies in immunohistochemistry?

Non-specific binding in immunohistochemistry with PPP1R16A antibodies can be addressed through several strategies:

• Optimize Blocking Conditions:

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C.

  • Test different blocking reagents: 5-10% normal serum from the same species as the secondary antibody, 3-5% BSA, or commercial blocking buffers.

  • Include 0.1-0.3% Triton X-100 in the blocking solution to reduce hydrophobic interactions.

• Antibody Dilution and Incubation:

  • Further dilute primary antibody if background persists (try series: 1:200, 1:500, 1:1000).

  • Extend primary antibody incubation to overnight at 4°C rather than 1-2 hours at room temperature.

  • Add 0.1-0.5% Tween-20 to antibody diluent to reduce non-specific binding.

• Antigen Retrieval Optimization:

  • Compare heat-induced epitope retrieval using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0).

  • Adjust retrieval time (10-30 minutes) to find optimal conditions.

  • Allow slides to cool slowly to room temperature after retrieval.

• Additional Controls and Treatments:

  • Include an absorption control by pre-incubating antibody with excess immunizing peptide.

  • If endogenous peroxidase activity is an issue, treat sections with 0.3-3% hydrogen peroxide before blocking.

  • For tissues with high endogenous biotin, use a biotin-streptavidin blocking kit.

  • If autofluorescence is a problem, treat sections with 0.1-1% Sudan Black B or commercial autofluorescence quenchers.

• Secondary Antibody Considerations:

  • Use highly cross-adsorbed secondary antibodies specific to the host species of the primary antibody.

  • Dilute secondary antibodies further (1:500-1:2000) and ensure short incubation times (30-60 minutes).

The A47452 antibody has been validated for IHC applications and may serve as a good starting point, as it demonstrates reactivity with human, mouse, and rat samples .

What strategies can address weak or absent signals when detecting PPP1R16A by Western blotting?

When facing weak or absent signals in Western blotting for PPP1R16A, consider these troubleshooting strategies:

• Sample Preparation Improvements:

  • Increase protein concentration (load 50-75 μg total protein).

  • Use fresh samples or minimize freeze-thaw cycles.

  • Add phosphatase inhibitors to preserve potential phosphorylation sites.

  • Try different lysis buffers: RIPA buffer for general extraction, NP-40 for milder conditions, or specific membrane protein extraction kits.

• Transfer and Membrane Optimization:

  • Extend transfer time or reduce voltage to improve transfer of higher molecular weight proteins.

  • Use PVDF membranes (0.45 μm pore size) rather than nitrocellulose for potentially better protein retention.

  • Verify transfer efficiency using reversible total protein stains.

• Antibody-Specific Adjustments:

  • Reduce antibody dilution (try 1:250-1:500 initially).

  • Extend primary antibody incubation to overnight at 4°C.

  • Try a different PPP1R16A antibody targeting an alternative epitope.

  • For polyclonal antibodies, later bleeds may have higher titer and affinity.

• Detection System Enhancement:

  • Switch to more sensitive detection systems (e.g., high-sensitivity ECL substrates).

  • Use signal enhancement systems such as biotin-streptavidin amplification.

  • Extend exposure times for chemiluminescence detection.

  • Consider alternative detection methods such as fluorescence-based systems.

• Confirming Expression Levels:

  • Verify PPP1R16A expression levels in your sample type using public databases.

  • Consider enrichment strategies if expression is low (e.g., immunoprecipitation before Western blotting).

  • Use positive control samples known to express PPP1R16A, such as pancreatic tissue or PPP1R16A-transfected 293T cells as demonstrated in published results .

Researchers have successfully detected PPP1R16A using ECL development techniques with antibody concentrations around 1 μg/mL, resulting in clear bands at the expected 58 kDa molecular weight .

How do post-translational modifications affect antibody recognition of PPP1R16A?

Post-translational modifications (PTMs) can significantly impact antibody recognition of PPP1R16A through several mechanisms:

• Impact on Epitope Accessibility:

  • Phosphorylation sites within or adjacent to antibody epitopes can block antibody binding.

  • Given PPP1R16A's role in phosphorylation regulation, it may itself be subject to regulatory phosphorylation events.

  • Conformational changes induced by PTMs may hide or expose different epitopes.

• Common PTMs Affecting PPP1R16A Detection:

  • Phosphorylation: As a regulatory subunit of protein phosphatase 1, PPP1R16A likely undergoes regulatory phosphorylation.

  • Ubiquitination: May affect protein stability and detection in Western blotting.

  • Prenylation: PPP1R16A (MYPT3) has been reported to undergo prenylation, which affects its membrane localization.

• Strategies to Address PTM Interference:

  • Use phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) in lysis buffers if detecting phosphorylated forms.

  • For ubiquitinated proteins, include deubiquitinase inhibitors (e.g., N-ethylmaleimide).

  • When studying membrane-associated forms, use appropriate detergent-based extraction methods.

  • Consider using multiple antibodies targeting different epitopes to provide comprehensive detection regardless of modification status.

• Verification Approaches:

  • Treat lysates with phosphatases before immunoblotting to determine if phosphorylation affects detection.

  • Use phosphorylation-specific antibodies when available to complement total protein detection.

  • Consider mass spectrometry analysis to map actual PTMs present in your experimental system.

• Selection of Appropriate Antibodies:

  • Review the epitope information provided by manufacturers to determine if the target sequence contains known or predicted modification sites.

  • For studying specific modified forms, select antibodies raised against the modified peptide sequence.

  • For total protein detection regardless of modification status, choose antibodies targeting regions less likely to be modified.

When working with PPP1R16A, researchers should consider its functional context as a regulatory protein in phosphorylation pathways, which suggests it may undergo dynamic modification in different cellular contexts .

How can PPP1R16A antibodies be used in functional studies of protein phosphatase 1 regulation?

PPP1R16A antibodies can serve as valuable tools in functional studies of protein phosphatase 1 (PP1) regulation through several methodological approaches:

• Co-Immunoprecipitation Studies:

  • Use PPP1R16A antibodies to pull down native protein complexes containing PP1 catalytic subunits.

  • Analyze composition of immunoprecipitated complexes by Western blotting or mass spectrometry.

  • Compare complex formation under different cellular conditions (e.g., quiescence vs. proliferation).

  • Protocol: Lyse cells in non-denaturing buffer (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5% glycerol), incubate with PPP1R16A antibody (2-5 μg) overnight at 4°C, add protein A/G beads for 2 hours, wash 4-5 times, and elute for analysis.

• Phosphatase Activity Assays:

  • Deplete PPP1R16A using antibodies in immunoprecipitation and measure resulting changes in PP1 activity.

  • Alternatively, add PPP1R16A antibodies directly to phosphatase reaction mixtures to determine if antibody binding affects enzymatic activity.

  • Assay PP1 activity using colorimetric substrates (e.g., p-nitrophenyl phosphate) or target-specific phosphopeptides with phosphorylation-specific detection methods.

• Subcellular Localization Studies:

  • Use immunofluorescence with PPP1R16A antibodies to track its localization and co-localization with PP1.

  • Monitor changes in localization in response to cellular stimuli.

  • Protocol: Fix cells in 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, block with 5% BSA, incubate with PPP1R16A antibody (1:100-1:500) overnight at 4°C, detect with fluorophore-conjugated secondary antibody, and counter-stain nuclei with DAPI.

• Conformational Change Analysis:

  • Use epitope-specific antibodies to detect conformational changes in PPP1R16A upon binding to PP1 or other interactors.

  • Apply techniques such as limited proteolysis followed by epitope mapping with different antibodies.

• Functional Blocking Studies:

  • Microinject PPP1R16A antibodies into cells to acutely block protein function.

  • Monitor subsequent changes in PP1 substrate phosphorylation (e.g., myosin light chain) using phospho-specific antibodies.

These approaches leverage the specificity of validated PPP1R16A antibodies to interrogate the functional role of this regulatory subunit in controlling PP1 activity toward various substrates, particularly phosphorylase, myosin light chain, and myosin, which are known to be regulated by PPP1R16A .

What are the best practices for using PPP1R16A antibodies in immunofluorescence studies?

Optimizing immunofluorescence studies with PPP1R16A antibodies requires attention to several best practices:

• Fixation and Permeabilization Optimization:

  • Test multiple fixation methods: 4% paraformaldehyde (10-15 minutes) preserves structure while maintaining antigen accessibility; methanol fixation (-20°C, 10 minutes) may better expose certain epitopes.

  • Optimize permeabilization: 0.1-0.5% Triton X-100 (5-10 minutes) for nuclear/cytoplasmic staining; milder detergents like 0.1% saponin for membrane proteins.

  • For PPP1R16A, which may associate with membranes through prenylation, careful optimization of these parameters is essential to preserve localization while enabling antibody access.

• Antibody Selection and Validation:

  • Choose antibodies validated specifically for immunofluorescence applications.

  • The A47452 antibody has been validated for IHC applications and may be suitable for immunofluorescence .

  • Perform peptide competition assays to confirm specificity.

  • Include both positive control cells known to express PPP1R16A and negative controls (either low-expressing cells or siRNA-treated samples).

• Signal Optimization Protocol:

  • Block thoroughly with 5-10% normal serum (from secondary antibody host species) for 1 hour at room temperature.

  • Use primary antibody at optimized dilutions (typically start at 1:100-1:500) and incubate overnight at 4°C.

  • Apply fluorophore-conjugated secondary antibodies at 1:500-1:1000 for 1 hour at room temperature (protect from light).

  • Include DAPI (1:1000) for nuclear counterstaining.

  • Mount with anti-fade mounting medium to prevent photobleaching.

• Co-localization Studies:

  • For co-localization with PP1 catalytic subunits, use antibodies from different host species to enable simultaneous detection.

  • Include appropriate controls for spectral bleed-through.

  • Analyze co-localization using software that calculates overlap coefficients (e.g., Pearson's or Manders' coefficients).

• Advanced Imaging Considerations:

  • Use confocal microscopy for precise subcellular localization.

  • Consider super-resolution techniques (STED, STORM, SIM) for detailed analysis of PPP1R16A distribution.

  • Capture z-stacks to ensure complete visualization of 3D distribution.

  • For quantitative analysis, maintain consistent exposure settings across samples.

• Troubleshooting Common Issues:

  • High background: Increase blocking time, dilute antibodies further, add 0.1% Tween-20 to washings.

  • Weak signal: Decrease antibody dilution, extend incubation time, try alternative fixation methods.

  • Punctate artifacts: Filter antibody solutions, ensure complete permeabilization, use fresher antibody aliquots.

By following these guidelines, researchers can optimize detection of PPP1R16A in immunofluorescence studies to reveal its subcellular distribution and potential co-localization with functional partners .

How can quantitative Western blotting be optimized for measuring changes in PPP1R16A expression levels?

Optimizing quantitative Western blotting for PPP1R16A expression analysis requires a systematic approach to ensure accuracy and reproducibility:

• Sample Preparation for Quantitative Analysis:

  • Standardize cell/tissue lysis procedures using consistent buffer composition and protein extraction methods.

  • Determine protein concentration using reliable methods (BCA or Bradford assay) with standard curves.

  • Prepare multiple aliquots of samples to avoid freeze-thaw cycles.

  • Create a standard curve using recombinant PPP1R16A protein or a well-characterized positive control sample.

• Gel Loading and Electrophoresis:

  • Load equal amounts of total protein (25-50 μg) as verified by Bradford/BCA assays.

  • Include a standard curve with 4-5 dilutions (e.g., 100%, 75%, 50%, 25%) of a reference sample.

  • Run duplicate or triplicate samples when possible.

  • Use pre-cast gels for consistency across experiments.

• Transfer and Validation:

  • Verify transfer efficiency using reversible total protein stains (Ponceau S or SYPRO Ruby).

  • Use PVDF membranes for better protein retention and quantitative linearity.

  • Consider stain-free technology or total protein normalization rather than single housekeeping proteins.

• Antibody Incubation and Detection:

  • Use validated PPP1R16A antibodies with demonstrated specificity at optimized concentrations.

  • Ensure that detection is in the linear range by performing preliminary experiments with different exposure times.

  • For fluorescence-based detection, use high-quality fluorophore-conjugated secondary antibodies.

  • For chemiluminescence, use enhanced reagents with consistent reaction times.

• Data Analysis and Normalization:

  • Use total protein normalization rather than single housekeeping genes when possible.

  • If using housekeeping proteins, validate their stability under your experimental conditions.

  • Analyze band intensity using software with background subtraction capabilities.

  • Express results as fold-change relative to controls rather than absolute values.

• Validation and Controls:

  • Include positive controls (PPP1R16A-transfected 293T cells have shown clear bands at 58 kDa) .

  • Use tissue samples with known expression levels (pancreas tissue has been used successfully) .

  • For verification of specificity, consider knockdown/knockout controls when available.

• Statistical Analysis:

  • Perform at least three independent biological replicates.

  • Use appropriate statistical tests (t-test, ANOVA) depending on experimental design.

  • Report both means and measures of variability (standard deviation or standard error).

Researchers have successfully used ECL development techniques with an antibody concentration of 1 μg/mL to detect PPP1R16A in Western blots of human pancreas lysate and PPP1R16A-transfected 293T cell lysate, showing clear bands at the expected 58 kDa size .

How can PPP1R16A antibodies contribute to understanding disease mechanisms?

PPP1R16A antibodies can provide valuable insights into disease mechanisms through several research applications:

• Expression Analysis in Disease States:

  • Compare PPP1R16A levels between normal and pathological tissues using immunohistochemistry and Western blotting.

  • Analyze tissue microarrays spanning multiple disease stages to correlate expression with disease progression.

  • Use PPP1R16A antibodies in conjunction with other markers to develop diagnostic or prognostic signatures.

  • Historical precedent: PPP1R16A has been investigated as a molecular marker in endometrial carcinoma, as noted in publications cited in antibody resources .

• Signaling Pathway Dysregulation:

  • Investigate PPP1R16A's role in signaling networks by studying its interactions with PP1 and downstream effects on substrate phosphorylation.

  • Use proximity ligation assays with PPP1R16A antibodies to visualize and quantify protein-protein interactions in situ.

  • Explore how alterations in PPP1R16A expression or localization affect cellular phosphorylation patterns in disease states.

• Therapeutic Target Validation:

  • Use antibodies to validate PPP1R16A as a potential therapeutic target by correlating its expression or activity with disease parameters.

  • Employ antibody-based approaches to modulate PPP1R16A function in cellular or animal models.

  • Screen for compounds that alter PPP1R16A-PP1 interactions and validate their effects using antibody-based detection methods.

• Biomarker Development:

  • Evaluate PPP1R16A as a diagnostic or prognostic biomarker using quantitative immunoassays.

  • Develop PPP1R16A-based liquid biopsy approaches for non-invasive disease monitoring.

  • Create multiplex assays incorporating PPP1R16A alongside other biomarkers for improved diagnostic accuracy.

• Functional Studies in Disease Models:

  • Track PPP1R16A localization changes in response to disease-relevant stimuli.

  • Combine PPP1R16A antibody staining with markers of cellular processes (proliferation, apoptosis, migration) to correlate its expression with functional outcomes.

  • Use in vivo imaging with labeled antibodies to monitor PPP1R16A in animal disease models.

PPP1R16A's role in regulating protein phosphatase 1 activity toward substrates including myosin light chain suggests potential involvement in diseases affecting muscle function, cell motility, and contractile processes . Additionally, its investigation as a molecular marker in endometrial carcinoma points to potential relevance in cancer biology .

What emerging techniques can be combined with PPP1R16A antibodies to advance protein phosphatase research?

Several cutting-edge techniques can be combined with PPP1R16A antibodies to drive innovations in protein phosphatase research:

• Proximity-Based Proteomics:

  • BioID or APEX2 proximity labeling: Fuse biotin ligase to PPP1R16A and use antibodies to verify expression before proteomics analysis of interacting partners.

  • Proximity Ligation Assay (PLA): Combine PPP1R16A antibodies with antibodies against PP1 catalytic subunits or potential substrates to visualize and quantify specific interactions in situ with nanometer resolution.

  • FRET-based interaction assays: Use fluorophore-conjugated PPP1R16A antibodies in combination with labeled PP1 antibodies to study dynamic interactions.

• Single-Cell Analysis Technologies:

  • Mass cytometry (CyTOF): Label PPP1R16A antibodies with metal isotopes for high-dimensional single-cell analysis in heterogeneous populations.

  • Single-cell Western blotting: Adapt PPP1R16A antibodies for microfluidic platforms that perform Western blotting on individual cells.

  • Imaging mass cytometry: Combine PPP1R16A antibodies with spatial proteomic approaches to map expression patterns in tissues with subcellular resolution.

• Advanced Microscopy Applications:

  • Super-resolution microscopy (STORM, PALM, STED): Optimize PPP1R16A antibodies for these techniques to visualize nanoscale distribution and co-localization patterns.

  • Expansion microscopy: Adapt immunostaining protocols for physically expanded samples to achieve super-resolution with standard microscopes.

  • Lattice light-sheet microscopy: Use fluorescently labeled PPP1R16A antibodies for high-speed 3D imaging of dynamic processes with minimal phototoxicity.

• Functional Genomics Integration:

  • CRISPR screens with antibody-based readouts: Use PPP1R16A antibodies to quantify protein levels or modifications following genome-wide CRISPR perturbations.

  • Antibody-based ChIP-seq or CUT&RUN: If PPP1R16A has nuclear functions, employ these techniques to map genomic binding sites.

  • Spatial transcriptomics combined with antibody staining: Correlate PPP1R16A protein expression with local transcriptional profiles.

• Therapeutic Development Applications:

  • Antibody-drug conjugates: Explore the potential of PPP1R16A antibodies for targeted delivery in relevant disease contexts.

  • Intrabodies: Develop cell-penetrating antibody fragments that can modulate PPP1R16A function intracellularly.

  • Nanobody development: Create camelid-derived single-domain antibodies against PPP1R16A for improved tissue penetration and novel applications.

These emerging techniques, when combined with well-validated PPP1R16A antibodies, have the potential to significantly advance our understanding of protein phosphatase regulation in normal physiology and disease states .

How can researchers evaluate and compare the performance of different commercial PPP1R16A antibodies?

Systematic evaluation of commercial PPP1R16A antibodies is essential for selecting the most appropriate reagent for specific research applications:

• Multi-Parameter Comparative Analysis Framework:

  • Create a structured testing pipeline evaluating each antibody across applications (WB, IHC, IF, ELISA).

  • Document key performance metrics: sensitivity (limit of detection), specificity (background/non-specific bands), reproducibility (inter-assay variation).

  • Develop a scoring system weighted for your specific application needs.

  Antibody	Reactivity	Applications	Host	Specificity Score	Sensitivity Score	Reproducibility Score
  A99952	Human	WB, ELISA	Rabbit	To be determined	To be determined	To be determined
  A47452	Human, Mouse, Rat	ELISA, WB, IHC	Rabbit	To be determined	To be determined	To be determined
  ab89135	Human	WB	Mouse	To be determined	To be determined	To be determined
  PACO22525	Human	ELISA, WB	Rabbit	To be determined	To be determined	To be determined

• Specificity Validation Experiments:

  • Side-by-side testing using positive controls (PPP1R16A-transfected cells) .

  • Validation in knockout/knockdown systems where available.

  • Peptide competition assays using the immunizing peptide.

  • Cross-reactivity assessment with related proteins (other PP1 regulatory subunits).

• Sensitivity and Dynamic Range Assessment:

  • Create standard curves with recombinant PPP1R16A protein.

  • Compare detection limits across antibodies using serial dilutions.

  • Evaluate linear dynamic range for quantitative applications.

  • Test sensitivity in detecting endogenous expression in low-expressing tissues/cells.

• Application-Specific Performance Metrics:

  • For Western blotting: Background, non-specific bands, signal-to-noise ratio.

  • For IHC/IF: Background staining, specificity of cellular/subcellular localization.

  • For IP: Pull-down efficiency, non-specific binding.

  • For ELISA: Standard curve characteristics, detection range, reproducibility.

• Lot-to-Lot Consistency Testing:

  • Compare multiple lots of the same antibody when available.

  • Maintain reference samples for comparative testing of new lots.

  • Document performance changes observed with new lots.

• Documentation and Reporting:

  • Create detailed protocols documenting optimal conditions for each antibody.

  • Record image data for all validation experiments with standardized acquisition settings.

  • Consider publishing or sharing validation data to benefit the research community.

Based on the available search results, researchers can start by evaluating rabbit polyclonal antibodies that demonstrate reactivity with their species of interest and are validated for their intended application. For human samples, multiple options are available, including A99952, A47452, PACO22525, and ab89135, while researchers working with mouse or rat samples might prioritize A47452, which has been validated for cross-reactivity with these species .

What are the current limitations in PPP1R16A antibody technology, and how might these be addressed in future research?

Current limitations in PPP1R16A antibody technology present several challenges that researchers should consider:

Addressing these limitations will require coordinated efforts between antibody developers, validation initiatives, and researchers to generate more comprehensive, well-characterized reagents for PPP1R16A research .

What are the recommended best practices for reporting PPP1R16A antibody usage in scientific publications?

Comprehensive reporting of PPP1R16A antibody usage in scientific publications is essential for reproducibility and appropriate interpretation of results:

• Detailed Antibody Identification Information:

  • Manufacturer name and location.

  • Complete catalog number (e.g., A47452, ab89135, PACO22525) .

  • Clone number for monoclonal antibodies or lot number for polyclonals.

  • RRID (Research Resource Identifier) when available.

  • Host species and antibody isotype.

• Validation Evidence:

  • Reference previous validation studies or include validation data in supplementary materials.

  • Describe controls used (positive controls, negative controls, peptide competition).

  • For critical findings, include validation using orthogonal methods (e.g., genetic knockdown).

  • If using for a novel application, include thorough validation data.

• Detailed Methodology:

  • Complete experimental protocols with antibody concentrations or dilutions (e.g., 1:500-1:3000 for WB) .

  • Buffer compositions and incubation conditions (time, temperature).

  • Detection methods with specific reagents and parameters.

  • Equipment settings and image acquisition parameters.

• Results Reporting:

  • Show representative full blots/images including molecular weight markers.

  • Indicate specific band size observed (expected ~58 kDa for PPP1R16A) .

  • For quantitative analyses, include details on normalization method.

  • Report replicate numbers and statistical analyses.

• Limitations and Considerations:

  • Acknowledge any limitations in antibody performance.

  • Address potential cross-reactivity issues with related proteins.

  • Discuss how antibody specificity might impact interpretation of results.

  • Note any inconsistencies with previous reports using different antibodies.

• Data Availability:

  • Include raw, unprocessed image data in supplementary materials or data repositories.

  • Provide access to analysis pipelines or custom scripts used for image quantification.

• Sample-Specific Information:

  • For tissue samples, include detailed information on preparation and fixation.

  • For cell lines, include authentication information and passage number.

  • Note any sample-specific optimizations of standard protocols.