PPP1R27 Antibody Protocol: IHC, WB & IF Applications

PPP1R27 Antibody is a research-grade immunological reagent targeting the protein phosphatase 1 regulatory subunit 27, enabling detection of PPP1R27 protein expression across multiple experimental platforms including Western blot, immunohistochemistry, and immunofluorescence.

Product SKU

Ab349605

Target Protein

PPP1R27 (Protein Phosphatase 1 Regulatory Subunit 27)

Reference Clone/Source

absea #OC-309

Recommended Applications

Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)

Storage Recommendation

Store at -20°C; avoid repeated freeze-thaw cycles

Overview of PPP1R27 and Antibody Specifications

PPP1R27 (Protein Phosphatase 1 Regulatory Subunit 27) is a member of the protein phosphatase 1 (PP1) regulatory subunit family. PP1 regulatory subunits modulate the substrate specificity, subcellular localization, and catalytic activity of the PP1 catalytic subunit, playing important roles in diverse cellular processes including cell cycle regulation, signal transduction, and metabolism.

The PPP1R27 Antibody (Aladdin SKU: Ab349605, reference clone absea #OC-309) is a research-grade reagent designed for the specific detection of PPP1R27 protein in experimental samples. This antibody is validated for use in Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) applications. Researchers should consult the product datasheet for confirmed species reactivity, host species, clonality, and recommended dilution ranges prior to beginning experiments.

Western Blot (WB) Protocol

Materials Required: SDS-PAGE gel, PVDF or nitrocellulose membrane, blocking buffer (5% non-fat milk or BSA in TBST), PPP1R27 Antibody (Ab349605), HRP-conjugated secondary antibody, ECL detection reagent.

1. Sample Preparation: Lyse cells or homogenize tissue in RIPA buffer supplemented with protease and phosphatase inhibitors. Determine protein concentration by BCA or Bradford assay. Prepare samples in Laemmli loading buffer and denature at 95°C for 5 minutes.
2. Gel Electrophoresis: Load 20–40 µg total protein per lane on an appropriate percentage SDS-PAGE gel. Run at 80–120 V until the dye front reaches the gel bottom.
3. Transfer: Transfer proteins to PVDF membrane (pre-activated in methanol) at 100 V for 60–90 minutes (wet transfer) or according to the semi-dry transfer system instructions.
4. Blocking: Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature with gentle agitation.
5. Primary Antibody Incubation: Dilute PPP1R27 Antibody (Ab349605) at the recommended dilution (refer to product datasheet) in blocking buffer. Incubate overnight at 4°C with gentle rocking.
6. Washing: Wash membrane 3 × 10 minutes in TBST.
7. Secondary Antibody: Incubate with appropriate HRP-conjugated secondary antibody (1:5000–1:10000) in blocking buffer for 1 hour at room temperature.
8. Detection: Wash 3 × 10 minutes in TBST, then apply ECL substrate and image using a chemiluminescence imaging system.

Note: Optimal antibody dilution and incubation conditions should be empirically determined for each experimental system.

Immunohistochemistry (IHC) Protocol

Materials Required: Formalin-fixed paraffin-embedded (FFPE) or frozen tissue sections, xylene, graded ethanol series, antigen retrieval buffer (citrate pH 6.0 or EDTA pH 9.0), hydrogen peroxide, blocking serum, PPP1R27 Antibody (Ab349605), biotinylated or polymer-based secondary detection system, DAB substrate, hematoxylin counterstain.

1. Deparaffinization and Rehydration (FFPE sections): Bake slides at 60°C for 30–60 minutes. Deparaffinize in xylene (2 × 5 minutes) and rehydrate through graded ethanol (100%, 95%, 70%) to distilled water.
2. Antigen Retrieval: Immerse slides in preheated citrate buffer (10 mM, pH 6.0) and heat in a pressure cooker or microwave for 10–20 minutes. Allow to cool for 20 minutes at room temperature.
3. Endogenous Peroxidase Blocking: Incubate sections in 3% H₂O₂ in methanol for 10 minutes. Wash with PBS (3 × 5 minutes).
4. Blocking: Apply 5–10% normal serum (matching secondary antibody host species) or commercial blocking reagent for 30–60 minutes at room temperature.
5. Primary Antibody: Apply PPP1R27 Antibody (Ab349605) diluted per datasheet recommendations in antibody diluent. Incubate overnight at 4°C in a humidified chamber.
6. Secondary Detection: Wash 3 × 5 minutes with PBS. Apply appropriate secondary antibody or polymer detection system for 30–60 minutes at room temperature.
7. Chromogenic Development: Apply DAB substrate for 5–10 minutes, monitor color development under microscope, and quench in water.
8. Counterstaining and Mounting: Counterstain with hematoxylin, dehydrate through graded ethanol, clear in xylene, and mount with permanent mounting medium.

Note: Antigen retrieval conditions and antibody dilution should be optimized for each tissue type and fixation protocol.

Immunofluorescence (IF) Protocol

Materials Required: Cells on coverslips or tissue sections, PBS, fixative (4% paraformaldehyde), permeabilization buffer (0.1–0.3% Triton X-100 in PBS), blocking buffer (5% BSA or normal serum in PBS), PPP1R27 Antibody (Ab349605), fluorophore-conjugated secondary antibody, DAPI nuclear stain, fluorescence mounting medium.

1. Fixation: Aspirate culture medium and wash cells with PBS. Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Wash 3 × 5 minutes with PBS.
2. Permeabilization: Incubate cells in 0.1–0.3% Triton X-100 in PBS for 10 minutes at room temperature. Wash 3 × 5 minutes with PBS.
3. Blocking: Incubate in blocking buffer (5% BSA or normal serum in PBS) for 30–60 minutes at room temperature.
4. Primary Antibody: Dilute PPP1R27 Antibody (Ab349605) at the recommended dilution in blocking buffer. Incubate overnight at 4°C in a humidified chamber protected from light.
5. Washing: Wash 3 × 5 minutes with PBS.
6. Secondary Antibody: Incubate with appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor series) at recommended dilution in blocking buffer for 1 hour at room temperature, protected from light.
7. Nuclear Counterstaining: Wash 3 × 5 minutes with PBS. Incubate with DAPI (0.5–1 µg/mL) for 5 minutes.
8. Mounting: Wash briefly, mount coverslips with anti-fade fluorescence mounting medium, and seal with nail varnish. Image using a fluorescence or confocal microscope.

Note: All steps after secondary antibody addition should be performed in subdued light to prevent photobleaching of fluorophores.

Troubleshooting and Tips

Optimizing antibody-based detection assays often requires systematic troubleshooting. The following guidance addresses common issues encountered when using PPP1R27 Antibody (Ab349605):

• No signal or weak signal: Verify that the antibody is reactive with the species of your experimental model. Check that antigen retrieval was performed correctly for IHC. Increase primary antibody concentration or extend incubation time. Ensure detection reagents and substrates are within expiry dates.
• High background: Increase blocking time or concentration. Optimize antibody dilution (use higher dilution). Extend washing steps. Ensure endogenous peroxidase is fully quenched in IHC applications.
• Non-specific bands in WB: Optimize antibody dilution. Try alternative blocking buffers (BSA instead of milk for phospho-targets, though PPP1R27 phosphorylation status should be confirmed). Run positive and negative controls in parallel.
• Inconsistent results between experiments: Standardize sample preparation, lysis, and protein quantification methods. Prepare antibody aliquots to avoid repeated freeze-thaw cycles. Use consistent incubation times and temperatures.
• Storage: Store PPP1R27 Antibody (Ab349605) at -20°C. Prepare working aliquots to minimize freeze-thaw cycles. Refer to the product datasheet for shelf-life information.

Frequently asked questions

What applications is the PPP1R27 Antibody (Ab349605) validated for?

The PPP1R27 Antibody (Ab349605) is recommended for use in Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) applications. Researchers should consult the product datasheet for confirmed application details, recommended dilutions, and species reactivity.

How should the PPP1R27 Antibody be stored to maintain activity?

PPP1R27 Antibody (Ab349605) should be stored at -20°C. To preserve antibody activity, it is recommended to prepare small working aliquots to avoid repeated freeze-thaw cycles. Always refer to the product datasheet for specific storage conditions and expiry information.

What controls should be included when using PPP1R27 Antibody in IHC or WB experiments?

It is strongly recommended to include a positive control (tissue or cell line known to express PPP1R27), a negative control (omitting the primary antibody), and where possible, an isotype control to assess non-specific background signal. These controls help validate staining specificity and ensure reliable data interpretation.

What is the recommended antigen retrieval method for PPP1R27 Antibody in IHC on FFPE sections?

Heat-induced epitope retrieval (HIER) using citrate buffer (10 mM, pH 6.0) is a standard starting point for most antibodies on FFPE sections. EDTA buffer (pH 9.0) is an alternative. The optimal antigen retrieval method for PPP1R27 Antibody (Ab349605) should be confirmed by consulting the product datasheet and may require empirical optimization for each tissue type.

References

1. Product datasheet: PPP1R27 Antibody, Aladdin Scientific, SKU Ab349605 (absea #OC-309). Please refer to the current product datasheet for validated application data.
2. Bollen M, Peti W, Ragusa MJ, Beullens M. The extended PP1 toolkit: designed to create specificity. Trends Biochem Sci. 2010;35(8):450-458. doi:10.1016/j.tibs.2010.03.002
3. Egloff MP, Johnson DF, Moorhead G, et al. Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1. EMBO J. 1997;16(8):1876-1887.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.