PPIL4 Antibody

What is PPIL4 and why is it studied in research?

PPIL4 (Peptidylprolyl Isomerase Cyclophilin-Like 4) is a protein encoded by the PPIL4 gene in humans (Gene ID: 85313) . It functions as a peptidylprolyl isomerase, facilitating protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds. The protein has a calculated molecular weight of approximately 57 kDa, though it is typically observed at 58-65 kDa in experimental conditions . PPIL4 is studied for its potential roles in RNA processing, protein folding, and cellular signaling pathways. Research on PPIL4 contributes to our understanding of fundamental cellular processes and may have implications for various pathological conditions where protein folding and RNA processing are disrupted.

What applications are PPIL4 antibodies typically used for?

PPIL4 antibodies are utilized across multiple experimental applications, with the most common being:

The optimal application depends on your specific research question, with Western blotting being the most widely validated method for PPIL4 detection in experimental systems .

What cell lines have been validated for PPIL4 antibody detection?

Multiple cell lines have been experimentally validated for PPIL4 detection using available antibodies:

• HEK-293 cells (human embryonic kidney cells)

• HCT 116 cells (human colorectal carcinoma)

• HeLa cells (human cervical cancer)

• HepG2 cells (human liver cancer)

• U-2 OS cells (human osteosarcoma)

These cell lines consistently show PPIL4 expression and have been used as positive controls in various experimental settings. When establishing a new experimental system, these cell lines can serve as reliable positive controls for antibody validation .

How do I differentiate between specific and non-specific binding when using PPIL4 antibodies?

Differentiating specific from non-specific binding requires implementing multiple validation approaches:

• Molecular weight verification: PPIL4 has a calculated molecular weight of 57 kDa but is typically observed between 58-65 kDa on Western blots . Any bands significantly outside this range may represent non-specific binding.

• Positive and negative controls: Include lysates from cells known to express PPIL4 (e.g., HeLa or HEK-293) as positive controls . For negative controls, consider using:

  • Lysates from cells where PPIL4 has been knocked down via RNAi

  • Pre-incubation of the antibody with immunizing peptide (blocking peptide)

  • Isotype control antibodies matching your primary antibody

• Cross-validation with multiple antibodies: Using antibodies targeting different epitopes of PPIL4 can confirm specificity. Available options include antibodies targeting:

  • AA 395-466 region (C-terminal)

  • AA 1-172 region (N-terminal)

  • AA 301-492 region

  • AA 122-151 region

• Immunogen comparison: Compare your experimental results with the specific immunogen sequence used to generate your antibody. For example, the sequence "EKEDEDYMPI KNTNQDIYRE MGFGHYEEEE SCWEKQKSEK RDRTQNRSRS RSRERDGHYS NSHKSKYQTD LY" is the immunogen for one monoclonal antibody .

What are the optimal conditions for detecting PPIL4 in various tissue types?

Detecting PPIL4 across tissue types requires optimization based on expression levels and tissue characteristics:

• Sample preparation:

  • For mouse kidney tissue (validated source ): Complete protease inhibitor cocktail is essential during homogenization

  • For cell lines: Lysis in RIPA buffer supplemented with phosphatase inhibitors improves detection

  • For human tissues: Antigen retrieval methods may need adjustment based on fixation method

• Antibody selection based on tissue:

  • For human tissues: Polyclonal antibodies show broader reactivity across tissue types

  • For mouse tissues: Specific rabbit polyclonal antibodies have been validated

  • For rat tissues: Limited validation data exists, but some antibodies show cross-reactivity

• Protocol modifications:

  • Extended blocking times (1-2 hours) may be necessary for tissues with high background

  • Secondary antibody concentration may need reduction (1:10,000 dilution) for tissues with high non-specific binding

  • For difficult tissues, overnight primary antibody incubation at 4°C improves specific signal

• Detection systems:

  • ECL-based methods provide sufficient sensitivity for most applications

  • For low-expression tissues, consider amplification systems or fluorescent-based detection

What are the critical parameters for reproducible immunofluorescence results with PPIL4 antibodies?

Achieving reproducible immunofluorescence results with PPIL4 antibodies depends on several critical parameters:

• Fixation method:

  • Paraformaldehyde (4%) for 15 minutes at room temperature preserves epitope accessibility

  • Methanol fixation (-20°C, 10 minutes) may enhance detection of certain epitopes

  • Avoid over-fixation which can mask epitopes

• Antibody dilution optimization:

  • Start with manufacturer-recommended dilutions (typically 1:200-1:800)

  • Perform dilution series (1:100, 1:200, 1:400, 1:800) to determine optimal signal-to-noise ratio

  • U-2 OS cells have been validated with 1:100 dilution for clear subcellular localization

• Permeabilization protocol:

  • 0.1-0.3% Triton X-100 for 5-10 minutes is typically sufficient

  • Saponin (0.1%) provides gentler permeabilization for some epitopes

  • Inadequate permeabilization is a common cause of false negatives

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval may be necessary for some fixation methods

  • Citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) are common retrieval solutions

  • Optimization of retrieval time is critical (typically 10-20 minutes)

• Controls and validation:

  • Nuclear counterstaining with DAPI to assess cell morphology and fixation quality

  • Peptide competition controls to confirm specificity

  • Secondary-only controls to establish background fluorescence levels

What is the optimal protocol for Western blot detection of PPIL4?

The following protocol has been optimized for reliable PPIL4 detection by Western blotting:

• Sample preparation:

  • Lyse cells in RIPA buffer with protease inhibitors

  • Use 25 μg protein per lane for cell lines with moderate to high PPIL4 expression

  • Denature samples at 95°C for 5 minutes in Laemmli buffer

• Gel electrophoresis and transfer:

  • 10% SDS-PAGE gel provides optimal resolution for 58-65 kDa PPIL4

  • Transfer to PVDF membrane at 100V for 60-90 minutes or 30V overnight at 4°C

  • Verify transfer with reversible Ponceau S staining

• Blocking and antibody incubation:

  • Block with 3-5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with primary antibody at 1:1000 dilution overnight at 4°C

  • Wash 3x10 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody at 1:10,000 for 1 hour

• Detection and imaging:

  • Develop using ECL substrate with 10-second exposure as starting point

  • Expected band size: 58-65 kDa

• Validation controls:

  • Positive controls: HEK-293, HCT 116, HeLa, or HepG2 cell lysates

  • Loading control: beta-actin, GAPDH, or alpha-tubulin

How can I optimize immunoprecipitation experiments with PPIL4 antibodies?

Successful immunoprecipitation of PPIL4 requires careful optimization:

• Lysate preparation:

  • Use 1.0-3.0 mg total protein for optimal results

  • NP-40 or RIPA buffer with protease inhibitors are suitable

  • Pre-clear lysate with Protein A/G beads (30 minutes at 4°C) to reduce non-specific binding

• Antibody binding:

  • Use 0.5-4.0 μg antibody per IP reaction

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

  • HeLa cell lysates have been validated for successful PPIL4 immunoprecipitation

• Bead selection and binding:

  • For rabbit host antibodies: Protein A or Protein A/G mix beads

  • For mouse host antibodies: Protein G beads

  • Add pre-washed beads and incubate 1-4 hours at 4°C

• Washing and elution:

  • Wash 3-5 times with cold lysis buffer

  • Additional high-salt wash can reduce non-specific binding

  • Elute with Laemmli buffer at 95°C for 5 minutes

• Controls:

  • Input control (5-10% of lysate used for IP)

  • IgG control from same species as primary antibody

  • IP-Western validation using a different PPIL4 antibody targeting another epitope

What approaches can resolve inconsistent results when working with PPIL4 antibodies?

When facing inconsistent results with PPIL4 antibodies, systematic troubleshooting approaches can help identify and resolve issues:

• Antibody validation and storage issues:

  • Perform antibody titration to determine optimal concentration for your specific sample

  • Avoid repeated freeze-thaw cycles that can reduce antibody activity

  • Store according to manufacturer recommendations (typically -20°C, with glycerol)

  • Aliquot antibodies upon receipt to prevent degradation

• Sample-specific considerations:

  • Protein degradation: Ensure complete protease inhibitor cocktail use

  • Post-translational modifications: Consider phosphatase inhibitors if phosphorylation affects epitope recognition

  • Expression levels: Different cell lines show variable PPIL4 expression; HeLa and HEK-293 are reliable positive controls

• Technical modifications:

  • For weak signals: Extended primary antibody incubation (overnight at 4°C)

  • For high background: Increase blocking time and washing steps

  • For multiple bands: Try reducing agent concentration adjustment or different antibody targeting another epitope

• Cross-validation approaches:

  • Use multiple antibodies targeting different PPIL4 epitopes

  • Combine detection methods (e.g., validate WB results with IF)

  • Consider RNA-level validation (qPCR) to confirm protein-level observations

• Application-specific adjustments:

  • For WB: Optimize transfer conditions for 58-65 kDa proteins

  • For IF: Try different fixation methods if cellular localization is unclear

  • For IP: Increase antibody amount within recommended range (0.5-4.0 μg)

How can PPIL4 antibodies be utilized in RNA processing research?

PPIL4 has potential roles in RNA processing, making its antibodies valuable tools in this research area:

• Co-immunoprecipitation studies:

  • IP with PPIL4 antibodies followed by mass spectrometry can identify novel RNA processing partners

  • Recommended antibody amount: 2-4 μg per IP reaction

  • Pre-treatment with RNase can distinguish RNA-dependent from direct protein interactions

• RNA immunoprecipitation (RIP):

  • Cross-link RNA-protein complexes with formaldehyde or UV

  • Immunoprecipitate with PPIL4 antibodies (1-4 μg per reaction)

  • Extract and analyze bound RNAs by sequencing or qPCR

  • Include appropriate controls (IgG, input RNA)

• Immunofluorescence co-localization:

  • Use PPIL4 antibodies at 1:200-1:400 dilution

  • Co-stain with known RNA processing markers

  • Analyze using high-resolution confocal microscopy

  • Quantify co-localization using appropriate statistical methods

• Cellular fractionation validation:

  • Separate nuclear, nucleolar, and cytoplasmic fractions

  • Validate fractionation using compartment-specific markers

  • Detect PPIL4 distribution across fractions by Western blotting

  • Use 1:500-1:1000 antibody dilution for optimal detection

• RNA processing under stress conditions:

  • Expose cells to transcriptional inhibitors or stress conditions

  • Monitor PPIL4 redistribution using immunofluorescence

  • Compare results across cell types with different PPIL4 expression levels

What considerations are important when using PPIL4 antibodies across different species?

When working with PPIL4 antibodies across species, several important considerations must be addressed:

• Validated species reactivity:

  • Human: Most antibodies show strong reactivity

  • Mouse: Several antibodies validated, particularly for kidney tissue

  • Rat: Limited validation but some cross-reactivity reported

  • Other species: Minimal validation data available

• Epitope conservation analysis:

  • Compare PPIL4 sequences across target species

  • Antibodies targeting highly conserved regions offer better cross-species reactivity

  • C-terminal region (AA 395-466) shows variation across species, potentially affecting antibody binding

• Application-specific validation:

  • Western blot: Start with 25 μg protein per lane for initial cross-species testing

  • Immunohistochemistry: May require species-specific optimization of antigen retrieval methods

  • Immunofluorescence: Fixation protocols may need adjustment based on species and tissue type

• Control selection:

  • Include known positive samples from the validated species alongside experimental samples

  • Consider species-specific positive controls (e.g., mouse kidney tissue for mouse samples)

  • When testing new species, include gradient loading to determine detection thresholds

• Protocol modifications:

  • Primary antibody concentration may need adjustment (typically 1.5-2× higher for non-validated species)

  • Extended incubation times may improve detection in non-validated species

  • Secondary antibody selection should match the host species of primary antibody

What are the best practices for quantitative analysis of PPIL4 using antibody-based methods?

For reliable quantitative analysis of PPIL4 using antibody-based methods, follow these best practices:

• Western blot quantification:

  • Use gradient loading to establish linear detection range

  • Include recombinant standards when absolute quantification is needed

  • Normalize to multiple housekeeping proteins (e.g., GAPDH, β-actin)

  • Use fluorescent secondary antibodies rather than chemiluminescence for wider linear range

  • Perform at least three biological replicates with consistent loading amounts (25 μg recommended)

• Immunofluorescence quantification:

  • Standardize image acquisition parameters (exposure, gain, offset)

  • Include fluorescence standards for calibration

  • Analyze multiple fields (>5) and cells (>50) per condition

  • Use appropriate software for unbiased quantification

  • Control for cell size and morphology variations

• ELISA development:

  • Use purified recombinant PPIL4 to generate standard curves

  • Optimize antibody concentrations (capture and detection)

  • Validate specificity using knockout or knockdown samples

  • Assess matrix effects from different sample types

  • Determine limits of detection and quantification

• Normalization strategies:

  • For cell-based assays: Normalize to cell number or total protein

  • For tissue analysis: Consider section thickness and area measurement

  • For Western blots: Total protein staining (e.g., Ponceau S) may provide better normalization than single reference proteins

• Statistical considerations:

  • Power analysis to determine appropriate sample sizes

  • Account for technical and biological variation

  • Apply appropriate statistical tests based on data distribution

  • Consider batch effects in multi-experiment analyses

How can PPIL4 antibodies be incorporated into advanced imaging techniques?

PPIL4 antibodies can be integrated into several advanced imaging approaches:

• Super-resolution microscopy:

  • STED or STORM microscopy can resolve PPIL4 subcellular localization beyond diffraction limit

  • Use higher primary antibody concentrations (1:100-1:200) for optimal signal

  • Secondary antibodies conjugated to photostable fluorophores are essential

  • Include fiducial markers for drift correction

• Live-cell imaging approaches:

  • For indirect visualization: Express PPIL4 with small epitope tags (FLAG, HA)

  • Use fluorescently labeled nanobodies against tags for live imaging

  • Validate with fixed-cell immunofluorescence using PPIL4 antibodies

  • Consider photobleaching concerns for long-term imaging

• Proximity ligation assay (PLA):

  • Combine PPIL4 antibodies with antibodies against potential interaction partners

  • Select antibodies from different host species (e.g., rabbit anti-PPIL4 with mouse anti-partner)

  • Optimize antibody dilutions specifically for PLA (typically higher concentrations than standard IF)

  • Include appropriate controls (single antibody, non-interacting protein pairs)

• Correlative light and electron microscopy (CLEM):

  • Immunogold labeling with PPIL4 antibodies for electron microscopy

  • Correlate with fluorescence microscopy data

  • Requires specialized sample preparation and high-specificity antibodies

  • Consider pre-embedding vs. post-embedding labeling approaches

• Tissue clearing and 3D imaging:

  • Optimize antibody penetration in cleared tissue samples

  • Extended incubation times (48-72 hours) with higher antibody concentrations

  • Validate with conventional thin-section immunofluorescence

  • Account for increased background in thick samples

What considerations are important when studying PPIL4 in primary cells versus cell lines?

Studying PPIL4 in primary cells introduces several considerations distinct from established cell lines:

• Expression level variations:

  • Primary cells typically show lower PPIL4 expression than immortalized lines

  • Increase protein loading (40-50 μg) for Western blot detection

  • Use more sensitive detection methods (ECL Prime or fluorescent secondaries)

  • Consider signal amplification systems for immunofluorescence

• Antibody validation strategy:

  • Always include positive control cell lines (HeLa, HEK-293) alongside primary cells

  • Validate primary cell-specific findings with multiple antibodies targeting different epitopes

  • Consider genetic approaches (siRNA) to confirm specificity in primary cells

  • Account for donor-to-donor variability in primary human cells

• Protocol adaptations:

  • Primary cell fixation may require gentler conditions (2% PFA rather than 4%)

  • Reduce detergent concentration for permeabilization (0.1% Triton X-100)

  • Extended blocking times to reduce background (2 hours minimum)

  • Longer primary antibody incubation (overnight at 4°C) for Western blot and immunofluorescence

• Primary cell-specific controls:

  • Age-matched samples for primary cells from different donors

  • Passage-matched cells for experiments over time

  • Tissue-specific positive and negative controls

  • Consider the impact of isolation methods on protein expression and epitope accessibility

• Technical challenges:

  • Limited material availability may necessitate micro-scale protocols

  • Increased heterogeneity requires larger sample sizes for statistical power

  • Sensitivity to culture conditions may affect PPIL4 expression or localization

  • Shorter experimental windows due to limited passage potential

What are the current limitations of PPIL4 antibodies and how might they be addressed?

Current PPIL4 antibodies present several limitations that researchers should be aware of:

• Epitope coverage limitations:

  • Most available antibodies target C-terminal or middle regions

  • Limited availability of N-terminal targeting antibodies

  • Future development of antibodies targeting functionally important domains would enhance research capabilities

  • Custom antibody generation may be necessary for specific epitopes

• Specificity concerns:

  • Cross-reactivity with related cyclophilin family proteins has been reported

  • Validation across multiple applications remains incomplete for many antibodies

  • Future approaches should include systematic validation with PPIL4 knockout controls

  • Comparative analysis of multiple antibodies is recommended for critical experiments

• Application restrictions:

  • Limited validation for immunohistochemistry on formalin-fixed tissues

  • Minimal data on chromatin immunoprecipitation applications

  • Future validation efforts should expand application range

  • Community-based validation reporting would accelerate progress

• Species reactivity limitations:

  • Strong validation primarily in human systems

  • Limited validation in model organisms beyond mouse

  • Future development should include broader species validation

  • Epitope mapping across species would aid antibody selection

• Technical improvements needed:

  • Development of monoclonal antibodies for better lot-to-lot consistency

  • Creation of application-specific antibodies (e.g., ChIP-grade, IHC-specific)

  • Conjugated versions for multi-parameter flow cytometry

  • Nanobody or recombinant antibody approaches for enhanced reproducibility

How might PPIL4 antibodies contribute to understanding disease mechanisms?

PPIL4 antibodies can significantly contribute to disease mechanism research in several areas:

• RNA processing disorders:

  • PPIL4 antibodies can help characterize aberrant splicing mechanisms

  • Immunoprecipitation followed by RNA-seq can identify disease-specific RNA interactions

  • Tissue microarray analysis can assess PPIL4 expression changes across disease states

  • Recommended approach: Combine 1:500 dilution Western blot with RNA-protein interaction studies

• Cancer research applications:

  • Expression profiling across tumor types (currently validated in several cancer cell lines)

  • Correlation of subcellular localization with disease progression

  • Analysis of post-translational modifications in cancer contexts

  • Patient-derived xenograft models can be analyzed with human-specific PPIL4 antibodies

• Neurodegenerative disease research:

  • Protein aggregation studies may benefit from PPIL4 antibodies

  • Co-localization with disease-specific protein aggregates

  • Analysis of stress granule association during cellular stress

  • Brain region-specific expression analysis requires careful antibody validation

• Developmental biology:

  • Tracking PPIL4 expression during differentiation processes

  • Analysis of protein-protein interaction networks during development

  • Combined with lineage markers for tissue-specific expression patterns

  • Knockout models can be validated using existing antibodies

• Therapeutic development:

  • Target engagement studies for compounds affecting PPIL4 function

  • Biomarker development for diseases with altered PPIL4 expression

  • Cellular assays for high-throughput screening of PPIL4 modulators

  • Proximity-based assays to monitor drug-induced conformational changes