PTN Antibody, FITC conjugated

What is Pleiotrophin (PTN) and why is it significant in research?

Pleiotrophin (PTN) is an important developmental cytokine that demonstrates high expression during embryogenesis but exhibits limited expression in adult tissues, where it is predominantly restricted to the brain . Its significance in research stems from its involvement in multiple biological processes, including neuronal development and its correlation with pathological conditions such as brain ischemia and Parkinson's disease . Additionally, PTN has been identified as a key player in cancer biology, with elevated serum levels observed in various solid tumors and hematological malignancies like multiple myeloma . The protein mediates mitogenic, transforming, and angiogenic activities through indirect activation of the receptor ALK (anaplastic lymphoma kinase) via PTPRB . These multifaceted roles make PTN a critical target for understanding disease mechanisms and developing potential therapeutic interventions.

What are the main applications of PTN Antibody, FITC conjugated in research settings?

FITC-conjugated PTN antibodies are primarily utilized in fluorescence-based detection methods for investigating PTN expression and localization in various biological samples. While the product data indicates this specific antibody has been validated for ELISA applications , FITC conjugation enables additional applications including immunofluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting (FACS). In neuroscience research, these antibodies can help visualize PTN expression patterns in neural tissues under different conditions . For cancer research, particularly in studies of multiple myeloma, FITC-conjugated PTN antibodies can be valuable for investigating PTN's role in tumor growth and angiogenesis, including the assessment of PTN production by malignant plasma cells . The fluorescent properties of the conjugate allow researchers to track PTN-expressing cells and study their interactions with other cellular components in complex biological systems.

What are the structural and biochemical properties of the PTN Antibody, FITC conjugated?

The PTN Antibody, FITC conjugated is a polyclonal IgG antibody derived from rabbit hosts immunized with recombinant Human Pleiotrophin protein (amino acids 33-168) . The antibody specifically targets human PTN (UniProtID: P21246) and has been protein G purified to >95% purity . The conjugation with FITC (Fluorescein isothiocyanate) provides the antibody with fluorescent properties, having an excitation wavelength of approximately 495nm and an emission wavelength of 519nm, producing a characteristic green fluorescence . The antibody is supplied in liquid form, preserved in a buffer containing 50% Glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody stability during storage and use. The FITC molecule is chemically linked to primary amine groups on the antibody through a process that requires careful control of reaction conditions including pH, temperature, and protein concentration to achieve optimal labeling without compromising antibody function .

What are the optimal storage conditions for PTN Antibody, FITC conjugated?

For PTN Antibody, FITC conjugated, proper storage is critical to maintain both antibody integrity and fluorescence activity. Upon receipt, the conjugated antibody should be stored at -20°C or -80°C to preserve its reactivity and fluorescent properties . Repeated freeze-thaw cycles should be strictly avoided as they can lead to antibody denaturation, aggregation, and loss of fluorescence intensity . For working solutions that will be used within a short period, aliquoting the stock solution into smaller volumes before freezing is highly recommended to minimize freeze-thaw cycles. During experimental handling, the antibody should be kept protected from light to prevent photobleaching of the FITC fluorophore, which can significantly reduce signal intensity over time . Some researchers utilize amber tubes or wrap containers in aluminum foil to provide light protection. Additionally, when removing the antibody from frozen storage, thawing should be done gradually at 4°C rather than at room temperature to minimize potential damage to the antibody structure and conjugate stability.

How should researchers optimize the FITC conjugation process for PTN antibodies?

The optimization of FITC conjugation to PTN antibodies involves several critical parameters to achieve maximum labeling efficiency while preserving antibody functionality. Research indicates that optimal conjugation occurs when using a purified IgG antibody preparation (ideally obtained through DEAE Sephadex chromatography) with high-quality FITC reagent . Key parameters to optimize include:

• Reaction conditions: Maximal labeling is achieved at room temperature, pH 9.5, with an initial protein concentration of approximately 25 mg/ml . The reaction typically reaches completion within 30-60 minutes under these conditions .

• Antibody preparation: Before conjugation, the antibody must be purified to remove any proteins or primary amines that could compete for FITC binding . Buffers containing primary amines (e.g., Tris) or thiols should be avoided, and concentration/purification kits may be necessary for antibodies in suboptimal buffers .

• FITC-to-protein ratio: The ratio of FITC molecules to antibody molecules (F/P ratio) must be carefully controlled to avoid under- or over-labeling . Over-labeled antibodies may experience reduced binding capacity, while under-labeled antibodies will produce insufficient fluorescence signal.

• Post-conjugation purification: Following the conjugation reaction, gradient DEAE Sephadex chromatography is recommended to separate optimally labeled antibodies from under- and over-labeled proteins , ensuring a homogeneous preparation with consistent properties.

What controls should be included when using PTN Antibody, FITC conjugated in experimental designs?

When designing experiments with PTN Antibody, FITC conjugated, several controls are essential to ensure experimental validity and interpretable results:

• Isotype control: An FITC-conjugated rabbit IgG antibody with the same isotype but irrelevant specificity should be included to identify non-specific binding and establish background fluorescence levels .

• Autofluorescence control: Unstained samples should be analyzed to determine the inherent autofluorescence of the biological material, particularly important when working with tissues that naturally contain fluorescent compounds.

• Blocking control: Samples pre-incubated with unconjugated PTN antibody or recombinant PTN protein (amino acids 33-168) before adding the FITC-conjugated antibody can demonstrate binding specificity .

• Positive control: Samples known to express PTN, such as multiple myeloma cell lines (RPMI8226, U266) or tissues with confirmed PTN expression, should be included to validate antibody performance .

• Negative control: Tissues or cell lines with minimal PTN expression can help establish the detection threshold and confirm specificity.

• Cross-reactivity assessment: When appropriate, testing the antibody against related proteins like Midkine can help confirm target specificity, as PTN shares structural similarities with other heparin-binding growth factors .

How can PTN Antibody, FITC conjugated be used to investigate PTN's role in tumorigenesis and angiogenesis?

The FITC-conjugated PTN antibody offers sophisticated approaches for investigating PTN's involvement in tumor development and blood vessel formation. Research has shown that PTN is highly expressed by multiple myeloma (MM) cells and promotes tumor growth , making this antibody valuable for mechanistic studies. For investigating tumor angiogenesis, researchers can employ the antibody in co-culture systems where monocytes are exposed to PTN-producing tumor cells . Flow cytometric analysis using the FITC-conjugated antibody can track PTN binding to monocytes and their subsequent differentiation into vascular endothelial-like cells, a process that has been demonstrated in multiple myeloma models .

In vivo studies can utilize the antibody for detecting PTN expression in tumor samples and correlating expression levels with tumor progression and vascularization . Additionally, blocking experiments where the antibody is used to neutralize PTN function have demonstrated reduced growth and enhanced apoptosis of MM cell lines and freshly isolated bone marrow tumor cells from MM patients in vitro . For more complex analyses, dual immunofluorescence combining the FITC-conjugated PTN antibody with markers for vascular structures can illuminate the spatial relationship between PTN-expressing cells and developing blood vessels within the tumor microenvironment .

What methodologies are recommended for detecting PTN expression in clinical samples using FITC-conjugated antibodies?

For clinical sample analysis, several methodologies can be employed with FITC-conjugated PTN antibodies to achieve robust and reliable results:

• Flow cytometry: For bone marrow or peripheral blood samples from patients with hematological malignancies, flow cytometric analysis using FITC-conjugated PTN antibodies can quantify the proportion of PTN-expressing cells . This approach allows for simultaneous assessment of multiple markers, enabling the identification of specific cell populations expressing PTN.

• Fluorescence microscopy: For tissue biopsies, immunofluorescence techniques using the FITC-conjugated antibody can visualize the spatial distribution of PTN expression. This method is particularly valuable for assessing PTN localization in relation to other tissue structures or cell types .

• Confocal microscopy: For high-resolution analysis of PTN expression patterns, confocal microscopy with FITC-conjugated antibodies provides detailed information about subcellular localization and co-localization with other proteins of interest.

• Quantitative image analysis: Computer-assisted quantification of fluorescence intensity from images acquired using FITC-conjugated PTN antibodies allows for objective measurement of expression levels across different samples or experimental conditions.

When working with clinical samples, it's crucial to establish standardized protocols for sample preparation, antibody concentration, and imaging parameters to ensure consistency and comparability between specimens . Additionally, correlation of PTN expression with clinical parameters and disease progression can provide valuable insights into its potential as a biomarker or therapeutic target .

How can researchers troubleshoot non-specific binding or weak signals when using PTN Antibody, FITC conjugated?

When encountering issues with non-specific binding or weak fluorescence signals, researchers should consider the following troubleshooting approaches:

• Antibody titration: Determine the optimal antibody concentration by testing a range of dilutions. While too little antibody results in weak signals, excessive antibody can increase non-specific binding .

• Blocking optimization: Inadequate blocking is a common cause of non-specific binding. Experiment with different blocking agents (BSA, normal serum, commercial blocking buffers) and durations to identify optimal conditions for your specific sample type .

• Fluorophore degradation assessment: FITC is sensitive to photobleaching and pH changes. Verify the integrity of the conjugate by measuring the fluorescence spectrum, and ensure proper storage conditions are maintained . Minimize exposure to light during all handling steps.

• Signal amplification strategies: For samples with low PTN expression, consider secondary amplification methods such as tyramide signal amplification or using anti-FITC secondary antibodies conjugated to brighter fluorophores .

• Fixation optimization: Overfixation can mask epitopes while underfixation can result in poor morphology. Test different fixation protocols to determine the best conditions for preserving both PTN antigenicity and sample integrity .

• Autofluorescence reduction: Employ strategies such as treatment with sodium borohydride, Sudan Black B, or commercial autofluorescence quenchers, particularly when working with tissues known to have high autofluorescence (e.g., brain, liver) .

• Permeabilization assessment: For intracellular PTN detection, evaluate different permeabilization reagents and conditions to ensure adequate antibody access while maintaining sample integrity .

Systematic documentation of troubleshooting steps and outcomes will help identify the optimal conditions for specific experimental systems and facilitate reproducibility .

How are FITC-conjugated PTN antibodies being used to study PTN's role in neurological disorders?

FITC-conjugated PTN antibodies are becoming instrumental in investigating PTN's complex roles in neurological conditions. Research has established that PTN expression increases during neuronal development and in response to stresses such as brain ischemia and Parkinson's disease . In neurological research applications, these antibodies enable researchers to visualize and quantify PTN expression patterns in neural tissues under both normal and pathological conditions.

In studies of neurodegenerative diseases, FITC-conjugated PTN antibodies can be used for co-localization studies with markers of neuronal stress, inflammation, or specific neural cell populations affected by the disease process . For instance, in Parkinson's disease models, these antibodies could help identify relationships between PTN expression and dopaminergic neuron degeneration or microglial activation. Similarly, in stroke models, the antibodies can track temporal and spatial changes in PTN expression during both the acute injury phase and subsequent recovery periods .

Additionally, these antibodies can facilitate research into PTN's potential neuroprotective functions, helping to elucidate whether PTN upregulation in neurological disorders represents a compensatory protective mechanism or contributes to disease pathology. Flow cytometry with FITC-conjugated PTN antibodies can also be employed to isolate and characterize neural progenitor cells expressing PTN, furthering our understanding of its role in neural regeneration and repair processes .

What potential exists for using PTN antibodies in therapeutic applications, and how might FITC conjugation assist in development?

Based on current research, PTN antibodies show significant therapeutic potential, particularly in cancer treatment, with FITC conjugation playing a valuable role in the developmental pipeline. Studies have demonstrated that inhibition of PTN with polyclonal anti-PTN antibodies reduced growth and enhanced apoptosis of multiple myeloma cell lines and freshly isolated bone marrow tumor cells from MM patients in vitro . Moreover, these antibodies markedly suppressed MM growth in vivo using a SCID-hu murine model .

The FITC conjugation provides several advantages during therapeutic development:

• Target validation and biodistribution studies: FITC-conjugated antibodies allow researchers to visualize the binding specificity and tissue distribution of candidate therapeutic antibodies .

• Pharmacokinetic analysis: The fluorescent tag enables tracking of antibody clearance and tissue accumulation over time, providing crucial data for dosing strategies .

• Mechanism of action studies: FITC-labeled antibodies facilitate investigation of downstream effects following PTN inhibition, such as disruption of tumor angiogenesis through monocyte transdifferentiation into vascular endothelial cells .

• Patient stratification biomarker development: The antibodies can help identify patients with PTN-dependent tumors who might benefit most from anti-PTN therapies .

The limited expression of PTN in normal adult tissues, primarily restricted to the brain , suggests that targeting PTN might have fewer side effects than therapies targeting more broadly expressed proteins. This characteristic makes PTN an attractive therapeutic target, particularly for conditions where the blood-brain barrier remains intact, potentially limiting antibody access to normal PTN-expressing neural tissues .

How can researchers quantitatively assess the fluorescence-to-protein (F/P) ratio for FITC-conjugated PTN antibodies?

Accurate determination of the fluorescence-to-protein (F/P) ratio is critical for ensuring consistent performance of FITC-conjugated PTN antibodies across experiments. The F/P ratio represents the average number of FITC molecules conjugated to each antibody molecule and directly impacts detection sensitivity and specificity . Several quantitative methods can be employed:

• Spectrophotometric determination: The most common method involves measuring absorbance at both 280 nm (protein) and 495 nm (FITC) using a spectrophotometer. The F/P ratio can be calculated using the formula:

  $F/P = \frac{A_{495} \times DF}{195,000} \times \frac{MW_{antibody}}{A_{280} - (0.35 \times A_{495})}$

  Where DF is the dilution factor, 195,000 is the molar extinction coefficient of FITC at 495 nm, MW_antibody is the molecular weight of the antibody (typically ~150,000 for IgG), and 0.35 represents FITC's contribution to absorption at 280 nm .

• Fluorescence standard curve: Prepare a standard curve using free FITC of known concentrations and measure fluorescence intensity. In parallel, measure the fluorescence of the conjugated antibody and interpolate the amount of FITC from the standard curve. Divide by the protein concentration (determined separately) to obtain the F/P ratio .

• Size-exclusion HPLC: This method separates free FITC from conjugated antibody and can be used with a fluorescence detector to quantify both protein content and fluorescence, enabling calculation of the F/P ratio.

• Gradient DEAE Sephadex chromatography: This technique not only allows determination of the F/P ratio but also facilitates separation of optimally labeled antibodies from under- and over-labeled proteins, resulting in a more homogeneous preparation .

For most immunofluorescence applications, an optimal F/P ratio typically falls between 3:1 and 6:1. Ratios lower than 3:1 may result in insufficient fluorescence signal, while ratios higher than 6:1 can lead to self-quenching and reduced antibody activity due to modification of critical lysine residues in the antigen-binding regions .

What statistical approaches are recommended for analyzing data generated using FITC-conjugated PTN antibodies?

• Flow cytometric data analysis: When quantifying the proportion of cells expressing PTN, statistical analysis should include comparison of mean fluorescence intensity (MFI) values between experimental groups . For multiple treatment conditions, one-way ANOVA followed by appropriate post-hoc tests (e.g., Tukey's or Bonferroni) should be employed to account for multiple comparisons . Non-parametric alternatives such as Kruskal-Wallis tests may be necessary if normality assumptions are violated.

• Quantitative image analysis: For immunofluorescence microscopy data, integrated density measurements (combining area and intensity) rather than raw intensity values often provide more reliable quantification . Colocalization studies should employ established metrics such as Pearson's correlation coefficient or Manders' overlap coefficient rather than subjective visual assessment.

• Correlation with clinical parameters: When relating PTN expression to patient outcomes or disease status, appropriate regression models (linear, logistic, or Cox proportional hazards, depending on the outcome variable) should be utilized . Multiple testing correction (e.g., Benjamini-Hochberg procedure) is essential when examining associations with numerous clinical variables.

• Reproducibility considerations: Data should be collected from multiple independent experiments, with attention to both biological and technical replicates . Power analyses should be conducted a priori to ensure sufficient sample sizes for detecting biologically meaningful effects.

• Software tools: Specialized software packages for cytometry data (FlowJo, Cytobank) or image analysis (ImageJ, CellProfiler) provide standardized algorithms for quantification and statistical testing, increasing reliability and reproducibility .

How can researchers differentiate between specific PTN signals and potential artifacts when using FITC-conjugated antibodies?

Distinguishing genuine PTN signals from artifacts requires systematic validation approaches and careful experimental design:

• Antibody validation controls: Compare staining patterns obtained with different anti-PTN antibodies (polyclonal vs. monoclonal, different epitope targets) to confirm consistent localization patterns . Use blocking experiments with recombinant PTN protein to demonstrate specificity of the observed signals .

• Spectral analysis: FITC has a characteristic excitation/emission spectrum (excitation ~495nm, emission ~519nm) . Spectral imaging can confirm that observed fluorescence matches FITC's profile rather than autofluorescence, which typically has broader emission spectra.

• Negative controls: Include tissues or cells known to have minimal PTN expression based on complementary techniques like RT-PCR or Western blotting . Any signal detected in these samples suggests potential non-specific binding.

• Genetic controls: When available, compare staining between wild-type samples and those with genetic PTN knockdown or knockout to validate signal specificity .

• Complementary techniques: Validate FITC-conjugated antibody results with orthogonal methods such as in situ hybridization for PTN mRNA, unconjugated antibodies with different detection systems, or mass spectrometry-based protein identification .

• Photobleaching assessment: True FITC signals will photobleach with characteristic kinetics when exposed to intense illumination. Autofluorescence and some artifacts are often more photostable .

• Fixation artifacts: Compare different fixation methods to identify potential fixation-induced artifacts, which may appear as non-specific staining patterns not present across multiple fixation protocols .

By systematically addressing these considerations, researchers can increase confidence that observed signals genuinely represent PTN expression rather than technical artifacts or non-specific binding .

What are the considerations for multiplexing FITC-conjugated PTN antibodies with other fluorescent markers?

When designing multiplexed fluorescence experiments incorporating FITC-conjugated PTN antibodies with other fluorophores, researchers should address several critical considerations:

• Spectral compatibility: FITC has excitation and emission maxima at approximately 495nm and 519nm, respectively . When selecting additional fluorophores, ensure minimal spectral overlap to avoid bleed-through and false co-localization. Ideal partners include red-emitting fluorophores (e.g., Texas Red, Cy5) rather than yellow-emitting dyes (e.g., PE, TRITC) which have significant overlap with FITC .

• Compensation requirements: For flow cytometry applications, proper compensation is critical when FITC is combined with other fluorophores. Single-stained controls for each fluorophore are essential for accurate compensation matrix calculation .

• Antibody host species compatibility: When using multiple primary antibodies, select those raised in different host species to prevent cross-reactivity of secondary detection reagents. If antibodies from the same species must be used, consider direct conjugates or sequential staining protocols with blocking steps between antibody applications .

• Signal intensity balancing: FITC may not be as bright as newer generation fluorophores such as Alexa Fluor dyes. When multiplexing, adjust antibody concentrations to balance signal intensities across all channels, particularly for quantitative colocalization studies .

• Photostability differences: FITC is relatively prone to photobleaching compared to more stable fluorophores. In time-lapse imaging or experiments requiring extended exposure times, consider the differential bleaching rates when interpreting apparent changes in colocalization over time .

• Fixation and mounting considerations: Some mounting media contain anti-fade agents that may affect certain fluorophores differently. Test compatibility of your specific combination of fluorophores with intended fixation and mounting protocols .

• Imaging equipment limitations: Ensure your microscope or flow cytometer has appropriate filter sets or spectral detection capabilities to cleanly separate all fluorophores in your multiplex panel .

What are the current limitations of PTN Antibody, FITC conjugated, and potential future developments?

Current limitations of FITC-conjugated PTN antibodies include several technical challenges that affect their utility in certain research contexts. FITC is relatively sensitive to photobleaching compared to newer fluorophores, which can limit applications requiring extended imaging periods or high-intensity illumination . Additionally, FITC's quantum yield decreases at higher pH, potentially affecting sensitivity in certain buffer conditions . The polyclonal nature of many commercially available PTN antibodies also introduces batch-to-batch variability that can complicate standardization across studies .

Another significant limitation is the relatively narrow application range validated for some commercial offerings, with products like those described in the search results primarily validated for ELISA rather than a broader spectrum of applications such as immunohistochemistry, Western blotting, or immunoprecipitation . Furthermore, current anti-PTN antibodies may not distinguish between different PTN isoforms or post-translational modifications, potentially masking biologically significant differences in PTN variants .

Future developments likely to address these limitations include:

• Development of monoclonal antibodies with defined epitope specificity for consistent performance

• Conjugation with more photostable fluorophores like Alexa Fluor dyes to enhance imaging capabilities

• Creation of antibodies specific to PTN phosphorylation states or splice variants

• Validation across broader application ranges to expand utility

• Development of antibody fragments (Fab, scFv) for improved tissue penetration in imaging applications

• Integration with emerging super-resolution microscopy techniques for nanoscale localization studies

These advancements would significantly enhance the utility of anti-PTN antibodies in both basic research and potential clinical applications .

How might researchers integrate PTN Antibody, FITC conjugated into broader multi-omics research approaches?

Integration of FITC-conjugated PTN antibodies into multi-omics research strategies offers powerful opportunities for comprehensive understanding of PTN biology across different biological scales. Researchers can implement several approaches:

• Single-cell multi-omics: Combine flow cytometry using FITC-conjugated PTN antibodies with single-cell RNA sequencing to correlate PTN protein expression with transcriptomic profiles at the individual cell level . This approach can reveal heterogeneity in PTN expression and identify gene networks associated with high PTN-expressing cells in complex samples like tumors or developing tissues.

• Spatial transcriptomics integration: Overlay immunofluorescence imaging using FITC-conjugated PTN antibodies with spatial transcriptomics data to correlate PTN protein localization with gene expression patterns across tissue regions . This integration provides context about the microenvironment surrounding PTN-expressing cells.

• Proteogenomic correlation: Connect PTN antibody-based proteomics data with genomic and transcriptomic datasets to identify genetic variants or regulatory elements influencing PTN expression levels . This approach can uncover mechanisms controlling PTN expression in different physiological and pathological states.

• Functional genomics validation: Use FITC-conjugated PTN antibodies to assess protein-level changes following CRISPR-based genetic manipulations of PTN or related pathway components . This provides functional validation of genomic findings and helps establish causality in PTN-related phenotypes.

• Phospho-proteomics connection: Combine detection of total PTN using FITC-conjugated antibodies with phospho-proteomics to examine downstream signaling events activated by PTN in different cellular contexts . This integration helps map PTN-triggered signaling networks.

• Clinical multi-omics: Correlate PTN antibody-based measurements in patient samples with other omics data and clinical outcomes to identify biomarker signatures and potential therapeutic targets in diseases like multiple myeloma .