PTPA Mouse mAb Usage & Protocol Guide | Aladdin Scientific

PTPA Mouse mAb is a high-specificity monoclonal antibody raised in mouse against human PTPA (Protein Phosphatase 2A Phosphatase Activator), designed for reliable detection and functional studies of PTPA across multiple immunoassay platforms.

Product SKU

Ab349231

Antibody Type

Mouse Monoclonal Antibody (mAb)

Target Protein

PTPA (Protein Phosphatase 2A Phosphatase Activator)

Reference Clone

absea #KC-3857

Recommended Storage

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

Host Species

Mouse

Background: PTPA and Its Biological Significance

PTPA (Protein Phosphatase 2A Phosphatase Activator), also known as PP2A phosphatase activator or PTPA/PPP2R4, is a highly conserved protein that plays a critical role in regulating the activity and substrate specificity of Protein Phosphatase 2A (PP2A). PP2A is one of the major serine/threonine phosphatases in eukaryotic cells and is involved in fundamental cellular processes including cell cycle progression, apoptosis, and signal transduction.

PTPA functions as a peptidyl-prolyl isomerase and an ATPase-dependent activator of PP2A, facilitating the proper folding and activation of the PP2A catalytic subunit. Dysregulation of PTPA and the PP2A complex has been implicated in various pathological conditions, making PTPA an important research target in oncology and neurodegenerative disease studies.

The PTPA Mouse mAb (Ab349231) provides researchers with a validated tool to detect and study endogenous PTPA protein expression, localization, and interaction networks in human and compatible model systems.

Recommended Protocol for Western Blot (WB)

The following protocol serves as a general guideline for using PTPA Mouse mAb (Ab349231) in Western Blot applications. Optimization may be required depending on your specific cell or tissue system.

1. Sample Preparation: Lyse cells or homogenize tissue in RIPA buffer supplemented with protease and phosphatase inhibitors. Determine protein concentration using a BCA or Bradford assay. Load 20–40 µg of total protein per lane on an SDS-PAGE gel (recommended 10–12% polyacrylamide).
2. Electrophoresis & Transfer: Run the SDS-PAGE gel under denaturing conditions and transfer to a PVDF or nitrocellulose membrane using wet or semi-dry transfer systems.
3. Blocking: Block the membrane with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature to minimize non-specific binding.
4. Primary Antibody Incubation: Dilute PTPA Mouse mAb at the manufacturer-recommended dilution in blocking buffer. Incubate the membrane overnight at 4°C with gentle agitation. (Refer to the product datasheet for the validated dilution range; specific dilution factors are subject to review — see review_flags.)
5. Washing: Wash the membrane three times with TBST for 10 minutes each wash.
6. Secondary Antibody: Incubate with an appropriate HRP-conjugated anti-mouse secondary antibody for 1 hour at room temperature.
7. Detection: Wash three times with TBST and detect using an ECL chemiluminescence substrate. Image using X-ray film or a digital imaging system.

Expected Band Size: Refer to the Ab349231 product datasheet for the confirmed molecular weight of PTPA. Additional bands may indicate post-translational modifications or non-specific binding.

Additional Application Guidelines: IHC and IF

PTPA Mouse mAb (Ab349231) may be suitable for Immunohistochemistry (IHC) and Immunofluorescence (IF) applications. The following general guidelines apply; always validate conditions empirically for your experimental system.

Immunohistochemistry (IHC-P):

• Tissue Preparation: Use formalin-fixed, paraffin-embedded (FFPE) sections (4–5 µm). Deparaffinize and rehydrate through a graded alcohol series.
• Antigen Retrieval: Perform heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) as appropriate.
• Blocking: Block endogenous peroxidase activity with 3% H₂O₂ for 10 minutes. Apply serum-free blocking buffer for 30 minutes at room temperature.
• Primary Antibody: Incubate sections with diluted PTPA Mouse mAb overnight at 4°C in a humidified chamber. Refer to the product datasheet for validated dilution.
• Detection: Use a polymer-based HRP detection system followed by DAB chromogen and hematoxylin counterstain.

Immunofluorescence (IF):

• Cell Fixation: Fix cells in 4% paraformaldehyde for 15 minutes at room temperature. Permeabilize with 0.1–0.2% Triton X-100 in PBS for 10 minutes.
• Blocking: Block with 5% normal goat serum or 1% BSA in PBS for 1 hour.
• Primary Antibody: Incubate with diluted PTPA Mouse mAb for 1–2 hours at room temperature or overnight at 4°C.
• Secondary Antibody: Use a fluorophore-conjugated anti-mouse secondary antibody (e.g., Alexa Fluor 488 or 555). Counterstain nuclei with DAPI.
• Mounting & Imaging: Mount with an aqueous anti-fade mounting medium and image by confocal or epifluorescence microscopy.

Storage, Handling, and Quality Considerations

Proper storage and handling of PTPA Mouse mAb (Ab349231) is essential to maintain antibody activity and ensure reproducible experimental results.

• Storage Temperature: Store the antibody at -20°C for long-term storage. For frequent use, short-term storage at 4°C is acceptable for up to one month.
• Freeze-Thaw Cycles: Minimize repeated freeze-thaw cycles, as they can lead to antibody aggregation and loss of activity. Aliquoting upon first thaw is strongly recommended.
• Formulation: Refer to the product datasheet (Ab349231) for the exact formulation, including buffer composition and any preservatives or stabilizers present.
• Sterility: Handle under clean conditions. Avoid microbial contamination of the stock solution.
• Lot Consistency: Document the lot number used in each experiment. Verify performance when switching to a new lot, as minor batch-to-batch variation may occur.
• Expiry: Check the expiration date on the vial label. Do not use expired reagents, as antibody performance may be compromised.

When using this antibody, always include appropriate positive and negative controls to validate staining specificity and assay performance.

Troubleshooting Common Issues

The table below provides guidance for resolving common issues encountered when using PTPA Mouse mAb (Ab349231).

Issue	Possible Cause	Recommended Action
No signal detected	Antibody concentration too low; insufficient antigen retrieval; protein degradation in sample	Increase primary antibody concentration; optimize antigen retrieval conditions; use fresh samples with protease inhibitors
High background / non-specific bands	Insufficient blocking; antibody concentration too high; secondary antibody cross-reactivity	Increase blocking time or concentration; reduce primary antibody dilution; use species-specific secondary antibody
Weak or inconsistent signal	Suboptimal incubation conditions; degraded antibody from freeze-thaw	Incubate overnight at 4°C; aliquot antibody to prevent repeated freeze-thaw; verify ECL substrate freshness
Band at unexpected molecular weight	Post-translational modifications; protein complexes; non-specific binding	Consult datasheet for expected MW; include positive control lysate; verify gel conditions

For persistent technical issues, contact Aladdin Scientific technical support with your lot number, experimental conditions, and representative images.

Frequently asked questions

What applications is PTPA Mouse mAb (Ab349231) validated for?

PTPA Mouse mAb (Ab349231) is intended for immunodetection applications including Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF). Please consult the official product datasheet for the complete and up-to-date list of validated applications and recommended dilutions, as validation data may be updated over time.

What is the recommended storage condition for PTPA Mouse mAb to maintain its stability?

PTPA Mouse mAb (Ab349231) should be stored at -20°C for long-term stability. Repeated freeze-thaw cycles should be avoided, as they can cause antibody aggregation and reduce activity. It is strongly recommended to aliquot the antibody into single-use volumes upon first thaw. For short-term use, the antibody may be stored at 4°C for up to one month.

What positive control should I use when testing PTPA Mouse mAb by Western Blot?

For Western Blot, a whole-cell lysate from a human cell line known to express PTPA (Protein Phosphatase 2A Phosphatase Activator) is recommended as a positive control. Commonly used cell lines expressing PTPA include HeLa or HEK293; however, please verify PTPA expression levels in your chosen cell line using published literature or database resources prior to use.

How should I troubleshoot high background when using PTPA Mouse mAb in IHC?

High background in IHC is commonly caused by insufficient blocking, excessive primary antibody concentration, or incomplete quenching of endogenous peroxidase activity. To resolve this, extend the blocking step to at least 1 hour using serum-free blocking buffer, optimize the primary antibody dilution, and ensure thorough endogenous peroxidase quenching with 3% H₂O₂. Including a no-primary-antibody negative control is essential to assess background levels.

References

1. Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochemical Journal. 2001;353(3):417-439. doi:10.1042/bj3530417
2. Longin S, Jordens J, Martens E, et al. An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator. Biochemical Journal. 2004;380(Pt 1):111-119. doi:10.1042/BJ20031643
3. Absea Biotechnology. KC-3857 PTPA Antibody Product Information Sheet. Absea Biotechnology Ltd.

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