MPP1 Antibody, FITC conjugated

How does FITC conjugation affect MPP1 antibody performance in immunofluorescence applications?

FITC (Fluorescein isothiocyanate) conjugation provides direct visualization of MPP1 in fluorescence microscopy without requiring secondary antibodies. While no specific data on MPP1-FITC conjugated antibodies appears in the provided resources, similar conjugated proteins like Concanavalin A-FITC demonstrate that proper conjugation maintains binding specificity while enabling fluorescent detection . When working with FITC-conjugated MPP1 antibodies, researchers should be aware that FITC has optimal excitation at 495 nm and emission at 519 nm, requiring appropriate filter sets. Additionally, FITC is susceptible to photobleaching and pH sensitivity (optimal at pH 8.0), which may necessitate careful experimental planning including anti-fade mounting media and pH-controlled buffers to maximize signal stability and detection sensitivity.

What are the recommended sample preparation methods for optimal MPP1 antibody binding?

For optimal binding of MPP1 antibodies in immunohistochemistry applications, paraffin-embedded tissues should undergo proper antigen retrieval. Based on successful applications of the unconjugated MPP1 antibody (ab96255), standard heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes is recommended . For immunofluorescence applications with FITC-conjugated antibodies, cells should be fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1-0.5% Triton X-100 for 5-10 minutes. Blocking with 3-5% BSA or normal serum from the same species as the secondary antibody (if used) for 30-60 minutes helps minimize non-specific binding. These protocols may require optimization depending on your specific cell type or tissue.

What are the expected subcellular localization patterns for MPP1 in different cell types?

MPP1 demonstrates distinct subcellular localization patterns that vary depending on the cell type and cell cycle phase. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, MPP1 primarily localizes to a proportion of interphase nuclei . During metaphase, MPP1 redistributes throughout the cytoplasm and perichromatin mass . In later stages of cell division (telophase/anaphase), MPP1 localizes to the stem body and midzone of the midbody . Tissue-specific expression patterns reveal that MPP1 antibody demonstrates remarkable staining of specific cell subsets in the cerebellum, ovary, and testis tissues . In AML cell lines, MPP1 forms a protein complex with ABCC4 at the plasma membrane, which can be disrupted by specific peptides or compounds like Antimycin A .

How can I validate the specificity of a FITC-conjugated MPP1 antibody?

To validate specificity of FITC-conjugated MPP1 antibodies, employ multiple complementary approaches. First, conduct Western blot analysis using cell lysates known to express MPP1 (such as HeLa cells) with both unconjugated and FITC-conjugated antibodies to confirm they recognize the same protein band at the expected molecular weight (approximately 52 kDa) . Second, perform immunoprecipitation experiments followed by mass spectrometry to verify the target protein's identity. Third, use competitive binding assays with unconjugated MPP1 antibodies to demonstrate specific displacement of the FITC-conjugated antibody signal. Fourth, compare staining patterns with different MPP1 antibody clones. Finally, implement knockout/knockdown validation by comparing staining in MPP1-expressing cells versus MPP1-deficient controls, which represents the gold standard for antibody validation.

How can the interaction between MPP1 and ABCC4 be effectively studied using FITC-conjugated MPP1 antibodies?

The protein interaction between MPP1 and ABCC4 can be effectively studied using FITC-conjugated MPP1 antibodies through several advanced approaches. First, implement Förster Resonance Energy Transfer (FRET) analysis by combining FITC-conjugated MPP1 antibodies with ABCC4 antibodies conjugated to a compatible acceptor fluorophore (such as TRITC). When the proteins interact, energy transfer between the fluorophores will occur, providing direct visual evidence of their proximity. Second, conduct co-localization studies using confocal microscopy with FITC-conjugated MPP1 antibodies and alternatively labeled ABCC4 antibodies, quantifying overlap using Pearson's correlation coefficient or Manders' overlap coefficient. Third, perform live-cell imaging to track the dynamics of this interaction during drug treatment using FITC-conjugated MPP1 antibodies in cells expressing fluorescently-tagged ABCC4. Fourth, combine with proximity ligation assays (PLA) to visualize and quantify the interaction with higher sensitivity than conventional co-localization studies .

What factors should be considered when interpreting MPP1 expression data in relation to drug resistance in AML?

When interpreting MPP1 expression data in relation to drug resistance in AML, researchers should consider several critical factors. First, the co-expression level of ABCC4 is essential, as research demonstrates a high correlation (r = 0.84) between MPP1 and ABCC4 expression in pediatric AML, particularly in the FAB M7 subtype . Second, the integrity of the PDZ-binding motif in ABCC4 is crucial for the MPP1-ABCC4 interaction, as disruption of this motif prevents complex formation and alters drug resistance profiles . Third, the subcellular localization of MPP1 and ABCC4 must be evaluated, as their membrane co-localization is functionally significant for drug efflux activity. Fourth, consider the specific chemotherapeutic agents being studied, as resistance mechanisms may vary. Fifth, assess the presence of compounds that might disrupt the MPP1-ABCC4 interaction, such as Antimycin A, which has been shown to reverse drug resistance in AML cell lines and primary patient samples . Finally, account for potential heterogeneity in patient samples, as MPP1 expression varies among AML subtypes and may have differential prognostic significance.

How might FITC-conjugated MPP1 antibodies be incorporated into high-content screening approaches for drug discovery?

FITC-conjugated MPP1 antibodies can be strategically incorporated into high-content screening (HCS) approaches for drug discovery targeting the MPP1-ABCC4 interaction. First, establish an automated image-based assay using FITC-conjugated MPP1 antibodies to quantify membrane localization of MPP1 in fixed cells after compound treatment, as disruption of the MPP1-ABCC4 complex removes ABCC4 from the plasma membrane and increases drug sensitivity . Second, implement a FRET-based screening platform using FITC-MPP1 antibodies and complementary labeled ABCC4 antibodies to directly measure protein-protein interaction disruption. Third, develop a live-cell kinetic assay to monitor real-time changes in MPP1 localization during drug treatment. Fourth, combine with high-throughput flow cytometry to quantify both MPP1 expression levels and subcellular localization patterns in response to compound libraries. Fifth, integrate computational approaches to identify structural features of compounds that effectively disrupt the MPP1-ABCC4 interaction, using Antimycin A as a positive control since it has been identified as a small molecule that disrupts this protein complex .

What are the potential pitfalls in experimental design when studying MPP1 localization during cell cycle progression?

When studying MPP1 localization during cell cycle progression using FITC-conjugated antibodies, researchers should be aware of several potential pitfalls. First, cell synchronization methods may artificially alter MPP1 localization patterns; therefore, validation across multiple synchronization techniques or the use of live-cell imaging on asynchronous populations is recommended. Second, fixation artifacts can significantly impact the observed subcellular distribution of MPP1, particularly during mitosis when cellular architecture is dramatically reorganized. Compare multiple fixation protocols (paraformaldehyde, methanol, and combinations) to ensure consistent results. Third, antibody accessibility issues may arise during different cell cycle phases due to changes in nuclear envelope integrity or chromatin condensation; optimize permeabilization conditions for each phase. Fourth, potential confusion between different proteins named MPP1 (p55/EMP55 versus KIF20B) necessitates careful antibody validation and experimental controls. Fifth, MPP1's distinct localization patterns across cell cycle phases (nuclear in interphase, cytoplasmic in metaphase, and at the midbody during telophase/anaphase) require precise cell cycle staging using established markers (e.g., phospho-histone H3, cyclin B1) for accurate interpretation.

How can FITC-conjugated MPP1 antibodies be used to investigate the mechanism by which MPP1 regulates neutrophil polarity?

To investigate MPP1's role in neutrophil polarity regulation using FITC-conjugated antibodies, implement a multi-faceted experimental approach. First, conduct time-lapse confocal microscopy with FITC-conjugated MPP1 antibodies in neutrophil-like cell lines or primary neutrophils during polarization in response to chemoattractants, capturing the dynamic redistribution of MPP1. Second, perform co-localization studies with markers of neutrophil polarity including phosphorylated AKT1, as MPP1 regulates neutrophil polarization by regulating AKT1 phosphorylation through a mechanism independent of PIK3CG activity . Third, utilize FACS-based analysis with FITC-conjugated MPP1 antibodies to quantify expression levels before and after polarization stimuli. Fourth, combine with super-resolution microscopy techniques (STORM, PALM) to visualize nanoscale organization of MPP1 at the leading edge versus trailing edge of polarized neutrophils. Fifth, implement optogenetic approaches to spatiotemporally manipulate MPP1 localization while monitoring neutrophil polarization and migration. Sixth, use phospho-specific antibodies alongside FITC-conjugated MPP1 antibodies to determine if MPP1 phosphorylation status changes during polarization processes.

What are the optimal storage conditions for maintaining FITC-conjugated MPP1 antibody stability and performance?

To maintain optimal stability and performance of FITC-conjugated MPP1 antibodies, implement strict storage protocols based on fluorophore and protein preservation principles. Store the conjugated antibody at -20°C in small aliquots (10-50 μL) to minimize freeze-thaw cycles, as each cycle can reduce activity by 5-10%. Protect from light using amber vials or by wrapping containers in aluminum foil, as FITC is particularly susceptible to photobleaching. Add a protein stabilizer (e.g., 1% BSA) and preservative (e.g., 0.02% sodium azide) to maintain antibody integrity during storage. The optimal buffer composition includes PBS at slightly alkaline pH (7.4-8.0), as FITC fluorescence is pH-dependent and maximized at pH 8.0. For working dilutions, store at 4°C and use within 1-2 weeks. Monitor stability through periodic quality control testing comparing fresh aliquots to previously thawed samples using flow cytometry or microscopy to detect signal deterioration. For long-term archival storage (>1 year), consider lyophilization with cryoprotectants such as trehalose or sucrose to maintain activity.

How can I optimize FITC-conjugated MPP1 antibody dilutions for various applications?

Optimizing FITC-conjugated MPP1 antibody dilutions requires systematic titration across different applications while considering signal-to-noise ratio. For immunofluorescence microscopy, start with a dilution series (1:50, 1:100, 1:200, 1:500, 1:1000) on positive control samples (e.g., HeLa cells) , evaluating both signal intensity and background levels. For flow cytometry, a similar titration approach is recommended but typically requires more concentrated antibody (starting around 1:20-1:50) due to shorter incubation times. For each application, calculate the signal-to-background ratio by dividing the mean fluorescence intensity of positive staining by that of negative controls. The optimal dilution will provide the highest ratio rather than simply the strongest signal. Create a standardization curve for each new lot of antibody to ensure consistency across experiments. Consider that optimal dilutions may vary based on fixation method, with aldehyde fixatives often requiring more antibody than alcohol-based fixatives due to potential epitope masking. For multiplexed applications, test for spectral overlap and adjust antibody concentrations accordingly to prevent bleed-through between channels.

What controls should be implemented when using FITC-conjugated MPP1 antibodies in co-localization studies?

Rigorous controls are essential for reliable co-localization studies using FITC-conjugated MPP1 antibodies. First, include single-stained controls for each fluorophore to establish proper filter settings and correct for spectral bleed-through, particularly important when pairing FITC with fluorophores having overlapping emission spectra. Second, implement biological negative controls using MPP1-deficient cells (knockdown/knockout) to establish background staining levels. Third, incorporate antibody specificity controls including isotype controls (FITC-conjugated non-specific IgG of the same isotype) and pre-adsorption controls (pre-incubating the antibody with excess MPP1 recombinant protein). Fourth, use positive controls such as known MPP1-interacting proteins like ABCC4, which should show co-localization based on their documented physical interaction . Fifth, include technical controls such as reversed labeling (switching fluorophores between target proteins) to ensure co-localization is not fluorophore-dependent. Sixth, employ quantitative co-localization methods (Pearson's correlation, Manders' overlap coefficient) rather than relying solely on visual assessment. Finally, prepare samples with deliberate misalignment of fluorescence channels as negative co-localization controls.

How can FITC-conjugated MPP1 antibodies be effectively used in flow cytometry applications?

For effective flow cytometry applications with FITC-conjugated MPP1 antibodies, implement a comprehensive optimization protocol addressing fixation, permeabilization, and analysis parameters. Begin with proper sample preparation: for cell surface epitopes, use gentle fixation (0.5-2% paraformaldehyde); for intracellular MPP1, which has distinct localization patterns across cell cycle phases , use methanol or saponin-based permeabilization after fixation. Optimize antibody concentration through titration experiments (typically 1:20-1:200) to identify the dilution that provides the highest separation index between positive and negative populations. Include compensation controls when multiplexing with other fluorophores to correct for spillover, as FITC has broad emission that may overlap with PE and other fluorochromes. Utilize viability dyes to exclude dead cells, which often show non-specific antibody binding. For cell cycle analysis with MPP1, combine with DNA content dyes (PI, DAPI) in separate channels to correlate MPP1 expression with cell cycle phases, reflecting its dynamic localization during mitosis . Implement both fluorescence-minus-one (FMO) and isotype controls to establish gating boundaries accurately.

What strategies can be employed to minimize background when using FITC-conjugated MPP1 antibodies in tissue sections?

To minimize background when using FITC-conjugated MPP1 antibodies in tissue sections, implement a comprehensive strategy addressing multiple sources of non-specific signals. First, perform thorough blocking (5-10% normal serum from the same species as secondary antibody plus 1-3% BSA) for at least 1-2 hours at room temperature. Second, include an avidin-biotin blocking step if endogenous biotin may be present in tissues. Third, reduce autofluorescence through tissue pretreatment with Sudan Black B (0.1-0.3% in 70% ethanol) for 20 minutes, particularly important for tissues with high lipofuscin content like brain, where MPP1 staining in cerebellum has been observed . Fourth, if using formalin-fixed tissues, quench aldehydes with 0.1-0.3M glycine buffer prior to antibody incubation. Fifth, optimize tissue permeabilization carefully, as excessive detergent can increase background while insufficient permeabilization may prevent antibody access. Sixth, extend washing steps (4-6 washes of 10 minutes each) with 0.1% Tween-20 in PBS. Finally, use mounting media with anti-fade agents specific for FITC to prevent photobleaching and maintain optimal signal-to-noise ratio during imaging.

What experimental approaches can determine if MPP1 phosphorylation status affects its interaction with ABCC4?

To investigate how MPP1 phosphorylation impacts its interaction with ABCC4, implement a multi-faceted experimental strategy. First, perform co-immunoprecipitation experiments using FITC-conjugated MPP1 antibodies before and after treatment with phosphatase inhibitors or kinase activators/inhibitors to modulate phosphorylation states, followed by Western blot analysis for ABCC4. Second, develop a phosphorylation-state-specific MPP1 antibody panel (potentially including FITC conjugates) targeting known or predicted phosphorylation sites, particularly recognizing MPP1's reported phosphorylation during G2/M transition . Third, employ mass spectrometry analysis of immunoprecipitated MPP1-ABCC4 complexes to identify specific phosphorylation sites and their occupancy during complex formation. Fourth, create phosphomimetic and phospho-null MPP1 mutants (replacing phosphorylatable residues with aspartate/glutamate or alanine, respectively) and assess their ability to bind ABCC4 using fluorescence-based interaction assays. Fifth, implement FRET analysis between differentially labeled phospho-MPP1 and ABCC4 to visualize how phosphorylation status affects their molecular proximity in living cells. Sixth, combine with functional drug efflux assays to determine if phosphorylation-dependent changes in the MPP1-ABCC4 interaction alter chemotherapeutic resistance profiles.

How can FITC-conjugated MPP1 antibodies be used in conjunction with super-resolution microscopy techniques?

FITC-conjugated MPP1 antibodies can be effectively utilized with super-resolution microscopy techniques to reveal nanoscale organizational details beyond conventional microscopy limits. For Structured Illumination Microscopy (SIM), which provides approximately 100 nm resolution, FITC-MPP1 antibodies can be directly employed with standard immunofluorescence protocols to visualize the fine distribution of MPP1 at the plasma membrane and its co-organization with ABCC4 . For Stimulated Emission Depletion (STED) microscopy, offering 30-80 nm resolution, consider higher concentration of FITC-MPP1 antibodies (approximately 2x standard dilution) to compensate for the depletion process, while using mounting media with specialized anti-fade agents compatible with STED. For Single-Molecule Localization Microscopy (STORM/PALM) achieving 10-20 nm resolution, FITC may not be optimal due to its relatively poor photoswitching properties; consider custom-conjugating MPP1 antibodies with better photoswitching fluorophores like Alexa Fluor 647 while using FITC-conjugated versions for complementary conventional imaging. For all super-resolution applications, implement rigorous controls including resolution standards and quantitative validation of structures observed. Combine with multiplexed imaging to simultaneously visualize MPP1 interaction with binding partners like ABCC4, revealing spatial relationships at previously unattainable resolution.

What approaches can be used to study the dynamics of MPP1 localization in living cells?

To study the dynamics of MPP1 localization in living cells, implement complementary approaches that overcome the limitations of FITC-conjugated antibodies, which cannot penetrate intact cell membranes. First, generate expression constructs for MPP1-GFP (or other fluorescent protein) fusion proteins, validating that their localization matches endogenous MPP1 patterns observed with antibodies in fixed cells, particularly the characteristic cell cycle-dependent distribution (nuclear in interphase, cytoplasmic in metaphase, midbody in telophase/anaphase) . Second, employ CRISPR-Cas9 knock-in strategies to tag endogenous MPP1 with fluorescent proteins, maintaining natural expression levels and regulatory mechanisms. Third, use nanobody-based fluorescent labels that can enter living cells when conjugated to cell-penetrating peptides. Fourth, implement photoactivatable or photoconvertible fluorescent protein fusions with MPP1 for pulse-chase experiments tracking specific subpopulations of MPP1 over time. Fifth, combine with Fluorescence Recovery After Photobleaching (FRAP) or Fluorescence Loss In Photobleaching (FLIP) to measure MPP1 mobility and exchange rates between cellular compartments. Finally, implement optogenetic approaches to manipulate MPP1 localization while simultaneously monitoring downstream effects on cell functions like neutrophil polarization .

How might FITC-conjugated MPP1 antibodies be used to investigate MPP1's role in neutrophil responses to pathogens?

To investigate MPP1's role in neutrophil responses to pathogens using FITC-conjugated MPP1 antibodies, implement an integrated experimental approach focusing on dynamic processes and functional outcomes. First, establish live-cell imaging protocols combining membrane-permeable fluorescent MPP1 trackers with pathogen challenge assays, examining real-time redistribution of MPP1 during neutrophil activation, polarization, and phagocytosis, building on MPP1's known role in regulating neutrophil polarity . Second, develop quantitative immunofluorescence protocols with FITC-MPP1 antibodies in fixed neutrophils to capture MPP1 redistribution at different time points after pathogen exposure. Third, implement high-content imaging analysis to correlate MPP1 localization patterns with functional neutrophil responses including reactive oxygen species production, degranulation, and NET formation. Fourth, combine with phospho-flow cytometry to simultaneously assess MPP1 levels and activation of downstream signaling pathways, particularly AKT1 phosphorylation which is regulated by MPP1 . Fifth, employ MPP1 knockdown/knockout approaches in neutrophil-like cell lines with rescue experiments using wild-type or mutant MPP1 to establish causal relationships between MPP1 function and antimicrobial responses. Finally, develop ex vivo models using neutrophils from patients with infections to correlate MPP1 expression and localization patterns with clinical outcomes.