PPIL6 Antibody

What is PPIL6 and what cellular functions does it perform?

PPIL6 (peptidylprolyl isomerase-like 6) is a protein encoded by the PPIL6 gene (Gene ID: 285755) with a calculated molecular weight of approximately 35 kDa (311 amino acids) . Based on immunofluorescent staining studies in human cell lines like U-251 MG, PPIL6 has been observed to localize to the nucleus, cytosol, and Golgi apparatus, suggesting potential roles in protein folding, cellular trafficking, and signaling pathways . As a member of the cyclophilin family, it likely possesses peptidyl-prolyl cis-trans isomerase activity, which catalyzes the isomerization of peptide bonds at proline residues to facilitate protein folding . Current research indicates its involvement in protein quality control mechanisms rather than solely functioning as a structural protein.

How do I select the appropriate PPIL6 antibody for my experimental needs?

Selection of the appropriate PPIL6 antibody should be based on several technical considerations specific to your experimental design. First, determine the required species reactivity - available antibodies demonstrate reactivity with human samples , while some also react with mouse and rat samples . Second, consider the intended application - different PPIL6 antibodies are validated for specific techniques including Western Blot (WB), Immunocytochemistry/Immunofluorescence (IF/ICC), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunohistochemistry (IHC) . Third, assess antibody format requirements - options include unconjugated formats in PBS, with or without BSA, and various buffer compositions . Finally, evaluate clonality needs - both monoclonal (recombinant) and polyclonal options are available, with recombinant monoclonal antibodies offering superior batch-to-batch consistency for longitudinal studies .

What are the validated applications for PPIL6 antibodies in research?

PPIL6 antibodies have been validated for multiple research applications with specific recommended dilutions and protocols. The following table summarizes the validated applications across different antibody products:

How should I optimize PPIL6 antibody concentration for Western blot analysis?

Optimization of PPIL6 antibody concentration for Western blot requires a systematic titration approach. Begin with the manufacturer's recommended dilution range (1:500-1:2400 for polyclonal or 0.04-0.4 μg/ml for monoclonal antibodies) . Perform initial validation using positive control lysates such as HeLa or HepG2 cells, which have been confirmed to express detectable levels of PPIL6 . Prepare a dilution series across the recommended range and run identical Western blots to identify the optimal signal-to-noise ratio. When analyzing the results, look for a clean band at approximately 30-35 kDa, which corresponds to the observed molecular weight of PPIL6 . If non-specific binding occurs, implement additional blocking steps using 5% non-fat milk or BSA, and consider incorporating wash steps with increased stringency (0.1-0.3% Tween-20 in TBS/PBS). For challenging samples, validation against PPIL6 overexpression lysates versus vector control lysates can establish definitive specificity, as demonstrated in previous characterization studies .

What are the critical parameters for successful immunofluorescence detection of PPIL6?

Successful immunofluorescence detection of PPIL6 requires attention to several critical parameters. Fixation method significantly impacts epitope accessibility - PFA/Triton X-100 fixation has been validated for optimal results . Begin with the recommended antibody dilution range (1:50-1:500) and optimize through titration experiments . Use established positive control cell lines such as HeLa, which demonstrate reproducible PPIL6 expression patterns . When analyzing subcellular localization, expect to observe signal in the nucleus, cytosol, and Golgi apparatus based on previous characterization in U-251 MG cells . For co-localization studies, pair with established Golgi markers (e.g., GM130), nuclear markers (e.g., DAPI), and appropriate cytosolic markers to confirm the distribution pattern. Implement technical controls including secondary-only controls to assess background and pre-adsorption controls to validate specificity. For optimal image acquisition, use confocal microscopy with appropriate channel settings to minimize bleed-through when performing multi-label experiments.

How do PPIL6 antibody storage conditions affect long-term performance?

PPIL6 antibody storage conditions significantly impact long-term stability and performance. Research-grade PPIL6 antibodies demonstrate different stability profiles depending on format and buffer composition. For short-term storage (up to 1 month), refrigeration at 4°C is acceptable for most formulations . For medium-term storage, most formulations should be maintained at -20°C, particularly those containing stabilizers like glycerol (typically 40-50%) . For optimal long-term preservation and retention of binding activity, some formulations specifically require ultra-low temperature storage at -80°C . The buffer composition significantly impacts stability - antibodies in PBS-only formulations (BSA and azide-free) are more susceptible to degradation and should be handled with particular care . To preserve activity during repeated use, aliquoting is essential to minimize freeze-thaw cycles, which can progressively degrade antibody performance . Each freeze-thaw cycle typically reduces activity by 5-10%, with noticeable performance decline after 5+ cycles. When transitioning between storage temperatures, allow gradual equilibration to minimize protein denaturation from thermal shock.

What factors contribute to inconsistent PPIL6 antibody performance across different experimental batches?

Multiple factors can contribute to inconsistent PPIL6 antibody performance across experimental batches. Antibody source represents a primary variable - polyclonal antibodies inherently show greater batch-to-batch variation compared to monoclonal or recombinant antibodies, which demonstrate superior consistency . Storage conditions significantly impact stability - deviations from recommended temperature ranges (-20°C to -80°C depending on formulation) or excessive freeze-thaw cycles can progressively degrade antibody activity . Sample preparation variables including protein extraction method, buffer composition, and protein denaturation conditions can affect epitope accessibility. Experimental protocol modifications (blocking reagents, incubation times, wash stringency) between batches introduce technical variability. For Western blot applications, transfer efficiency, membrane type, and detection chemistry selections represent additional variables. To systematically address inconsistency, implement a standard positive control (e.g., HeLa cell lysate) with each experiment as a reference point . For critical applications requiring absolute consistency, consider validated recombinant antibody options which offer "unrivalled batch-to-batch consistency, easy scale-up, and future security of supply" .

How can I distinguish specific from non-specific binding when using PPIL6 antibodies?

Distinguishing specific from non-specific binding when using PPIL6 antibodies requires implementing multiple validation strategies. First, perform parallel experiments with positive and negative control samples - HeLa and HepG2 cells serve as validated positive controls expressing detectable PPIL6 levels , while vector-only transfected HEK293T lysates can serve as negative controls . Second, verify signal corresponds to the expected molecular weight (~30-35 kDa for PPIL6) . Third, implement peptide competition assays using the immunogenic peptide sequence (such as the sequence KVVGLFSCPNFQIAKSAAENLKNNHPSKFEDPILVPLQEFAWHQYLQEKKRELKNETWEYSSSVISFVNGQFLGDALDLQKWA used for NBP1-88766) to demonstrate signal reduction. Fourth, compare staining patterns across antibodies raised against different epitopes of PPIL6 - concordant patterns suggest specific binding. Fifth, for immunofluorescence applications, compare subcellular localization to established patterns (nucleus, cytosol, and Golgi apparatus) . Sixth, perform PPIL6 knockdown or knockout studies to demonstrate signal reduction with decreased target expression. For systematic analysis, quantify signal-to-noise ratios and establish objective thresholds for distinguishing specific from background signals.

What are the most common technical issues when performing sandwich ELISA with matched PPIL6 antibody pairs?

When performing sandwich ELISA with matched PPIL6 antibody pairs such as the MP01635-2 system (84870-2-PBS capture and 84870-1-PBS detection) , several technical issues may arise. First, inadequate blocking can lead to high background signal - optimize blocking buffer composition (BSA vs. casein-based) and incubation parameters (time, temperature). Second, improper antibody concentration can result in suboptimal assay performance - perform checkerboard titration experiments with the capture and detection antibodies to identify optimal concentrations for maximizing signal-to-noise ratio. Third, matrix effects from complex biological samples can interfere with antigen-antibody interactions - prepare standard curves in matrix-matched diluent that mimics sample composition. Fourth, hook effects may occur at very high analyte concentrations - implement sample dilution strategies and ensure standard curve encompasses expected sample concentration range. Fifth, cross-reactivity with related proteins can compromise specificity - validate using recombinant PPIL6 protein versus related cyclophilin family members. Sixth, lot-to-lot variability of detection reagents (streptavidin-HRP, substrate) can affect signal intensity - maintain consistent reagent sources across experimental batches. Systematic troubleshooting should employ quantitative metrics including coefficient of variation analysis (target <15% for intra-assay, <20% for inter-assay), signal-to-noise ratio optimization, and limit of detection determination.

How can PPIL6 antibodies be effectively utilized in multiplex imaging and high-content screening applications?

PPIL6 antibodies can be effectively integrated into multiplex imaging and high-content screening workflows through several strategic approaches. For multiplex fluorescence imaging, utilize conjugation-ready formats such as 84870-1-PBS (BSA and azide-free) for custom labeling with spectrally distinct fluorophores that minimize bleed-through with other targets of interest. Implement panel design that accounts for PPIL6's known subcellular distribution across nucleus, cytosol, and Golgi apparatus by selecting complementary markers that enable comprehensive phenotypic profiling. For high-content screening applications, establish quantitative metrics based on PPIL6 localization patterns, expression levels, and colocalization coefficients with organelle markers. Develop robust image analysis pipelines incorporating machine learning algorithms for unbiased classification of cellular phenotypes based on PPIL6 distribution patterns. When designing multiplexed assays, account for potential antibody cross-reactivity by testing each antibody individually before combining into panels. Consider cyclic immunofluorescence or sequential imaging approaches for highly multiplexed studies to overcome spectral limitations. For optimal results in automated systems, standardize fixation and permeabilization protocols (PFA/Triton X-100) across sample preparation workflows to ensure consistent epitope accessibility.

What strategies can be employed to study PPIL6 protein-protein interactions using immunoprecipitation techniques?

Studying PPIL6 protein-protein interactions requires carefully designed immunoprecipitation strategies. Begin with antibody selection - for optimal results, use antibodies validated for immunoprecipitation applications with minimal cross-reactivity to other cyclophilin family members. Consider recombinant antibody formats for reproducible performance . Cell lysis conditions must be optimized to preserve physiologically relevant interactions - test multiple lysis buffers with varying detergent compositions (NP-40, Triton X-100, CHAPS) and stringency to identify conditions that maintain complex integrity while ensuring efficient extraction. For challenging interactions, implement crosslinking approaches using membrane-permeable crosslinkers (DSP, formaldehyde) prior to lysis. Co-immunoprecipitation experiments should include comprehensive controls: IgG-matched control immunoprecipitations, reciprocal pull-downs with antibodies against suspected interaction partners, and validation with overexpression systems (as previously demonstrated with PPIL6 overexpression in HEK293T cells) . For detecting transient or weak interactions, consider proximity labeling approaches using PPIL6 fusion constructs with BioID or APEX2. Analysis of immunoprecipitated complexes should employ sensitive detection methods, including Western blotting with validated antibodies against suspected partners and mass spectrometry for unbiased discovery of novel interactions.

How does post-translational modification status affect PPIL6 antibody recognition and experimental outcomes?

Post-translational modifications (PTMs) can significantly impact PPIL6 antibody recognition and experimental outcomes. PPIL6, like other cyclophilin family members, may undergo various PTMs including phosphorylation, ubiquitination, and SUMOylation that can alter epitope accessibility. When designing experiments, consider that antibodies raised against specific peptide regions may have differential recognition capabilities depending on the PTM status of those regions. For example, the immunogen sequence used for NBP1-88766 (KVVGLFSCPNFQIAKSAAENLKNNHPSKFEDPILVPLQEFAWHQYLQEKKRELKNETWEYSSSVISFVNGQFLGDALDLQKWA) contains multiple potential phosphorylation sites that could affect antibody binding if modified. To address this complexity, implement parallel detection strategies using antibodies recognizing distinct epitopes. For comprehensive analysis, combine immunological detection with mass spectrometry approaches to characterize the precise PTM landscape. When interpreting Western blot results showing multiple bands or unexpected molecular weights, consider the possibility of PTM-induced mobility shifts. For stimulation experiments investigating dynamic regulation of PPIL6, include appropriate time points and controls to capture the full spectrum of modification states. Additionally, when studying PPIL6 in different cellular contexts, account for tissue-specific or condition-dependent PTM patterns that may affect antibody recognition and experimental interpretation.

What considerations are important when using PPIL6 antibodies in tissue-specific expression analysis?

Tissue-specific expression analysis of PPIL6 using immunohistochemical approaches requires multiple methodological considerations. Begin with appropriate tissue preparation and antigen retrieval - HIER pH 6 retrieval has been specifically recommended for PPIL6 detection in paraffin-embedded sections . Antibody selection should consider validated reactivity across species of interest - while many PPIL6 antibodies have demonstrated human reactivity , some also show cross-reactivity with mouse and rat tissues , facilitating comparative studies. Establish appropriate controls for each tissue type - positive control tissues should include those with known PPIL6 expression, while negative controls should include both technical controls (primary antibody omission) and biological controls (tissues with minimal PPIL6 expression). When analyzing bronchial tissue specifically, expect "moderate membranous positivity in respiratory epithelial cells" based on previous characterization . For quantitative analysis, implement digital pathology approaches with annotated regions of interest and standardized scoring systems. Consider tissue-specific factors that may influence results including fixation variables, endogenous peroxidase activity, and autofluorescence. For multiplex tissue analysis, carefully design antibody panels accounting for species compatibility of primary antibodies and implement appropriate spectral unmixing for fluorescent detection systems.

How can PPIL6 antibodies be utilized in biomarker discovery and validation studies?

PPIL6 antibodies can be strategically deployed in biomarker discovery and validation studies through a systematic workflow. Initial discovery phases should employ antibodies validated for multiple applications (Western blot, IHC, IF) to characterize PPIL6 expression patterns across disease states and normal tissues . For quantitative assessment, develop standardized ELISA protocols using matched antibody pairs such as the MP01635-2 system (84870-2-PBS capture and 84870-1-PBS detection) , establishing reference ranges and analytical performance metrics. Transition to validation phases by implementing tissue microarray analysis with antibodies optimized for IHC-paraffin applications at established dilutions (1:50-1:200) . When evaluating PPIL6 as a potential biomarker in liquid biopsies, consider preanalytical variables including sample collection, processing time, and storage conditions that may affect protein stability. For multiplexed biomarker panels, utilize conjugation-ready antibody formats to enable simultaneous detection of PPIL6 alongside other candidate markers. When moving toward clinical application, implement analytical validation following CAP/CLIA guidelines, including assessment of precision, accuracy, analytical specificity, and reportable range. Throughout the validation process, maintain consistent antibody sources and standardized protocols to ensure reproducibility across research sites and experimental batches.

What are the critical considerations for using PPIL6 antibodies in comparative expression studies across different disease models?

When using PPIL6 antibodies for comparative expression studies across disease models, several critical considerations must be addressed. First, ensure antibody cross-reactivity validation across all species being studied - select antibodies with documented reactivity to human, mouse, and rat PPIL6 for translational research spanning multiple model systems . Second, standardize tissue preparation protocols across all experimental groups to minimize technical variables that could confound biological differences. Third, implement quantitative detection methods with appropriate dynamic range to capture both subtle and dramatic changes in PPIL6 expression. Fourth, design comprehensive controls including isotype controls, competitive binding controls, and disease-relevant positive and negative controls. Fifth, account for disease-specific factors that may impact antibody performance including altered protein folding, aggregation, or post-translational modifications in pathological states. Sixth, when comparing across models, normalize PPIL6 expression to appropriate housekeeping proteins validated for stability in the specific disease context. For robust statistical analysis, implement power calculations to determine appropriate sample sizes and apply appropriate statistical tests for both parametric and non-parametric data distributions. When interpreting results, contextualize findings within the broader molecular landscape of each disease model, accounting for species-specific differences in PPIL6 regulation and function.