PPP1R13L Antibody, FITC conjugated

What is the biological role of PPP1R13L in cellular pathways and why is it a significant target for antibody detection?

PPP1R13L (also known as iASPP) functions as a critical regulator in apoptosis and transcription pathways through its interactions with two key proteins:

• It binds to NF-kappa-B and inhibits its transcriptional activity

• It interacts with p53/TP53, potentially preventing associations between p53/TP53 and ASPP1 or ASPP2, thereby suppressing apoptosis activation

Additional biological functions include:

• Blocking HIV-1 virus transcription by inhibiting both NF-kappa-B and SP1 action

• Potentially acting as an oncoprotein, with overexpression accelerating tumor formation in RAS/E1A transformed cells

• Serving as a novel autophagy inhibitor in keratinocytes

This multifunctional nature makes PPP1R13L an important target for research in oncology, virology, and cell death pathways.

What are the optimal protocols for using FITC-conjugated PPP1R13L antibodies in immunofluorescence applications?

For optimal results with FITC-conjugated PPP1R13L antibodies in immunofluorescence applications:

Sample Preparation Protocol:

• Fix cells in Solution A fixative for 15 minutes at room temperature

• Wash in PBS buffer

• Permeabilize with 0.25% Triton X-100 for 15 minutes

• Wash in PBS buffer

• Add FITC-conjugated anti-PPP1R13L antibody at 1:50-200 dilution

• Incubate for 30 minutes at room temperature

• Wash in PBS buffer

• Counter-stain nuclei with DAPI (200 ng/ml)

• Mount and visualize using appropriate fluorescence microscopy settings for FITC detection

Optimization Notes:

• Signal intensity can be confirmed by flow cytometry using median fluorescence intensity (MFI) comparison to isotype controls

• When analyzing subcellular localization, co-staining with organelle markers (such as KDEL for ER, GOLGA2/GM130 for Golgi) may be helpful

• For tissue sections, antigen retrieval with TE buffer pH 9.0 is recommended

How can researchers troubleshoot non-specific binding or weak signals when using PPP1R13L antibodies in Western blotting?

When encountering issues with PPP1R13L antibody performance in Western blotting:

Troubleshooting Weak Signals:

• Adjust concentration: Use dilution ranges between 1:500-1:2000 for most applications

• Consider protein abundance: PPP1R13L is expressed predominantly in epithelial cells, skin, testis, heart, and stomach tissues

• Verify expected molecular weight: Look for bands approximately 100 kDa (isoform 1) and 50 kDa (isoform 2)

• Observed molecular weight may appear as 110 kDa in some cell types

Reducing Non-specific Binding:

• Use affinity-purified antibodies that have been validated against the specific epitope

• Include appropriate blocking agents (BSA or non-fat milk)

• Increase washing steps and duration

• Consider positive controls: HEK-293 cells and human heart tissue show reliable detection

For accurate controls, use proteasome inhibitor MG132 with PPP1R13L-overexpressing cells, which characteristically shows p53 accumulation .

What are the comparative advantages of using FITC-conjugated versus unconjugated PPP1R13L antibodies in different experimental contexts?

FITC-Conjugated PPP1R13L Antibodies:

Advantages:

• Direct detection without secondary antibody requirements

• Reduced protocol time and fewer washing steps

• Elimination of potential cross-reactivity from secondary antibodies

• Suitable for multicolor immunofluorescence when combined with other differently-conjugated primary antibodies

• Effective for flow cytometry applications with minimal background

Limitations:

• Fixed fluorophore with specific excitation/emission properties

• Cannot leverage signal amplification via secondary antibodies

• May have reduced sensitivity compared to enzyme-based detection systems

Unconjugated PPP1R13L Antibodies:

Advantages:

• Versatility across multiple detection methods (WB, ELISA, IHC, IF)

• Signal amplification possible through optimized secondary antibody systems

• Compatible with various detection methods (chemiluminescence, colorimetric)

• Generally longer shelf-life without photobleaching concerns

Limitations:

• Requires additional incubation steps and reagents

• Potential for cross-reactivity with secondary antibodies

Application-Specific Selection Guide:

• Choose FITC-conjugated for direct immunofluorescence, flow cytometry, or multi-color staining

• Select unconjugated for western blotting, where signal amplification is beneficial, or when greater flexibility in detection methods is needed

How can researchers effectively validate the specificity of PPP1R13L antibodies in their experimental systems?

Comprehensive Antibody Validation Strategy:

• Genetic Approaches:

  • Compare antibody reactivity in wild-type versus PPP1R13L knockdown/knockout cells

  • Documented cases show characteristic changes in autophagy markers (LC3B) when PPP1R13L/iASPP is knocked down in HaCaT or N-TERT cells

• Epitope Mapping Verification:

  • Verify antibody binding to the specific epitope region

  • For example, confirm whether the antibody targets amino acids 775-800 of human iASPP (isoform 1)

• Cross-Reactivity Assessment:

  • Test across multiple species (human, mouse, rat) if cross-reactivity is claimed

  • Perform peptide competition assays with the immunizing peptide

• Functional Validation:

  • In PPP1R13L-overexpressing cells, verify functional impact on p53 levels and NF-κB activity

  • Characteristic finding: PPP1R13L overexpression causes faster p53 degradation through proteasome pathway

• Application-Specific Controls:

  • For FITC-conjugated antibodies, include isotype control with matched FITC:protein ratio

  • For immunoprecipitation, perform reverse IP and validate with mass spectrometry

Key Expected Observations for Valid Antibodies:

• In Western blot: Bands at approximately 100 kDa and 50 kDa corresponding to isoforms 1 and 2

• In co-immunoprecipitation: Detection of PPP1R13L interactions with Atg5–Atg12 and Atg16L1 complexes

• In overexpression studies: Inhibition of p53-dependent apoptosis pathways

What are the key considerations for designing experiments to study PPP1R13L's role in apoptosis using antibody-based detection methods?

When designing experiments to investigate PPP1R13L's role in apoptosis:

Experimental Design Framework:

• Cell Model Selection:

  • Primary mouse embryonic fibroblasts (MEFs) transformed with HRAS and E1A provide well-characterized models with defined p53 status

  • Epithelial cell lines (HaCaT, N-TERT) are appropriate given PPP1R13L's predominant expression in epithelial tissues

• Manipulation Approaches:

  • Overexpression using retroviral vectors expressing PPP1R13L

  • Knockdown using specific shRNA constructs targeting PPP1R13L/iASPP

  • Caspase cleavage studies using anti-Fas antibody to trigger apoptosis

• Key Markers to Monitor:

  • p53 levels and activation status

  • NF-κB p65/RelA activity

  • Apoptotic markers (cleaved PARP, pro-caspase 8, pro-caspase 9)

  • Bcl-2 family proteins (Puma, Noxa, Mcl-1, Bim)

• Control Conditions:

  • Proteasome inhibitor MG132 treatment to assess protein degradation pathways

  • p53-null versus p53-wildtype conditions to distinguish p53-dependent and -independent effects

  • BrdU incorporation to monitor proliferation in parallel with apoptosis assessment

Specific Analytical Methods:

• Co-immunoprecipitation to detect PPP1R13L interactions with p53 and NF-κB

• Immunofluorescence to track subcellular localization during apoptosis induction

• Western blotting to monitor protein expression levels and cleavage products

• Flow cytometry with FITC-conjugated anti-PPP1R13L to quantify expression levels in apoptotic versus non-apoptotic populations

How does PPP1R13L expression correlate with tumor development, and what antibody-based approaches are most effective for studying this relationship?

PPP1R13L appears to have significant oncogenic properties based on several lines of research:

Expression Patterns in Cancer:

• Overexpression detected in eight human breast carcinomas expressing wild-type p53

• Elevated expression observed in certain leukemias

• Expression frequently upregulated in multiple human cancer types

Functional Impact on Tumorigenesis:

• Overexpression of PPP1R13L strongly accelerates tumor formation by RAS/E1A transformed cells

• Creates phenotype with multiple tumor nodes consistent with increased metastasis

• Modulates both p53-dependent and -independent apoptosis pathways

Optimal Antibody-Based Approaches:

• Tissue Microarray Analysis:

  • Use Anti-PPP1R13L antibodies at 1:50-1:500 dilution for IHC

  • Compare expression across tumor grades and correlate with p53 status

  • Recommend antigen retrieval with TE buffer pH 9.0 for optimal results

• In vivo Tumor Models:

  • Immunohistochemical staining of tumor xenografts derived from PPP1R13L-overexpressing cells

  • Correlate PPP1R13L expression with metastatic potential and tumor growth rate

• Mechanistic Studies:

  • Co-immunoprecipitation to evaluate PPP1R13L interaction with p53 in tumor samples

  • Western blotting to assess PPP1R13L-induced effects on p53 degradation

  • Analyze PPP1R13L's impact on autophagy inhibition in cancer cells using LC3B labeling

Research Applications Table:

What are the most effective protocols for using PPP1R13L antibodies in co-immunoprecipitation experiments to study protein-protein interactions?

For successful co-immunoprecipitation of PPP1R13L and its binding partners:

Optimized Co-IP Protocol:

• Cell Lysis:

  • Use lysis buffer containing: 1 M Tris, 2.5 M NaCl, 10% glycerol, 0.5 M glycerophosphate, 1% Tween-20, 0.5% Nonidet P-40, and EDTA-free complete protease inhibitor

  • Incubate for 15 minutes on ice

  • Clear lysates by centrifugation

• Immunoprecipitation:

  • Incubate 1 mg of cleared cell lysate with 1 μg of antibody against target protein (e.g., Atg5–Atg12 or Atg16L1)

  • Alternatively, use anti-PPP1R13L antibody for reverse co-IP

  • Incubate at 4°C overnight

• Precipitation and Washing:

  • Add 50 μl Protein-G–Sepharose beads

  • Incubate for 2 hours at 4°C

  • Precipitate and wash three times with NP-40 lysis buffer

• Detection:

  • Subject immunoprecipitates to SDS-PAGE

  • Perform Western blot analysis for PPP1R13L and interacting partners

Quantification Method:
For quantitative analysis of binding interactions, calculate the percentage of proteins in complex using densitometry values:

• Percentage of Atg5 bound to PPP1R13L/iASPP = [(Atg5 IP/Atg5 input)/(iASPP IP/iASPP input)×100]

• Percentage of Atg5 bound to Atg16L1 = [(Atg5 IP/Atg5 input)/(Atg16L1 IP/Atg16L1 input)×100]

Key Interacting Partners to Study:

• p53/TP53 (tumor suppressor)

• NF-κB p65/RelA (transcription factor)

• Atg5–Atg12 complex (autophagy machinery)

• Atg16L1 (autophagy machinery)

• ASPP1 or ASPP2 (apoptosis-stimulating proteins)

How do different fixation and antigen retrieval methods affect the performance of PPP1R13L antibodies in immunohistochemistry?

Fixation and antigen retrieval significantly impact PPP1R13L antibody performance in immunohistochemistry:

Fixation Methods Comparison:

Antigen Retrieval Optimization:

• Heat-Induced Epitope Retrieval (HIER):

  • TE buffer pH 9.0 (recommended primary method)

  • Citrate buffer pH 6.0 (alternative method)

  • Heat source options: microwave, pressure cooker, water bath

• Enzymatic Retrieval:

  • Generally less effective for PPP1R13L detection

  • May be tested if HIER methods fail

Critical Factors Affecting Performance:

• Epitope Accessibility:
  PPP1R13L antibodies targeting different regions (e.g., AA 775-800 vs. AA 83-102) may require different retrieval methods

• Tissue Type Considerations:

  • Human breast cancer tissue shows reliable PPP1R13L detection

  • Epithelial tissues generally show stronger signals due to higher endogenous expression

• Detection Systems:

  • DAB-based systems for brightfield microscopy

  • Fluorescent secondary antibodies for immunofluorescence

  • For FITC-conjugated antibodies, ensure fixation methods don't compromise fluorophore activity

Validation Approach:
Include a gradient of antibody dilutions (1:50, 1:100, 1:250, 1:500) and multiple antigen retrieval methods on serial sections of the same tissue to identify optimal conditions for each specific application.

What considerations should researchers take into account when studying the role of PPP1R13L in autophagy inhibition using antibody-based techniques?

When investigating PPP1R13L's role as an autophagy inhibitor:

Experimental Design Considerations:

• Cell Model Selection:

  • Keratinocytes (HaCaT, N-TERT) are well-validated models for studying PPP1R13L's impact on autophagy

  • Compare wildtype versus PPP1R13L/iASPP knockdown cells

• Autophagy Induction Methods:

  • Nutrient starvation (serum-free medium)

  • Chemical inducers (rapamycin, torin)

  • Differentiation-induced autophagy

• Key Markers to Monitor:

  • LC3B conversion (LC3-I to LC3-II) by Western blot

  • LC3B puncta formation by immunofluorescence

  • Autophagic flux by p62 degradation

  • Atg5–Atg12 complex formation

  • Atg16L1 interaction with PPP1R13L

Antibody-Based Methodologies:

• Immunofluorescence Protocol for LC3B Labeling:

  • Fix cells following treatment conditions

  • Permeabilize with 0.25% Triton X-100

  • Incubate with anti-LC3B antibody (0.25 μg)

  • Visualize with Alexa-Fluor-647-conjugated secondary antibody

  • Analyze median fluorescence intensity and puncta formation

• Co-immunoprecipitation for PPP1R13L-Autophagy Protein Interactions:

  • Immunoprecipitate endogenous Atg5–Atg12 and Atg16L1

  • Detect PPP1R13L co-precipitation by Western blot

  • Quantify interaction using densitometry

• Molecular Mechanisms to Investigate:

  • PPP1R13L competition with Atg16L1 for binding to Atg5–Atg12 complex

  • Impact on autophagosome formation

  • Effect on autophagic flux

Controls and Validation:

• Include rapamycin treatment as positive control for autophagy induction

• Monitor mTOR pathway activity (phospho-S6, phospho-4E-BP1, phospho-mTOR)

• Compare autophagy in differentiated versus undifferentiated cells

• Include Beclin-1 detection as additional autophagy marker

This detailed approach will enable researchers to comprehensively characterize PPP1R13L's role in autophagy regulation using antibody-based techniques.