ENTR1 Antibody, FITC conjugated

What is ENTR1 and what cellular processes does it regulate?

ENTR1 (Endosome Associated Trafficking Regulator 1) functions as a critical regulator of endosomal trafficking pathways. Research demonstrates that ENTR1 controls cell surface levels of receptors such as Fas and influences Fas-mediated apoptotic signaling . ENTR1 appears to be constitutively localized to endosomes, where it regulates the transition of cargo from early endosomes to the lysosomal compartment. Depletion of ENTR1 leads to impaired transit of proteins like Fas from early endosomal compartments to lysosomal compartments, resulting in increased cell surface expression of these proteins .

Studies have shown that while ENTR1 does not significantly co-localize with Fas under non-stimulated conditions (where Fas is primarily localized to the plasma membrane and ENTR1 to intracellular punctae), co-localization occurs as early as five minutes after stimulation with agonistic antibodies. This suggests that ENTR1 interacts with internalized receptors at the early endosome stage .

How does FITC conjugation to antibodies work on a molecular level?

FITC conjugation to antibodies occurs through a chemical reaction between the isothiocyanate reactive group (-N=C=S) of FITC and primary amines (-NH₂) present on antibody molecules, particularly on lysine residues and the N-terminal amino group. This reaction forms a stable thiourea bond between FITC and the antibody .

The conjugation reaction is highly dependent on several parameters:

• pH of the reaction buffer (optimal at pH 9.5)

• Temperature (room temperature is typically sufficient)

• Protein concentration (optimal at ~25 mg/ml)

• Reaction time (maximal labeling achieved in 30-60 minutes)

• Purity of both the antibody and FITC reagent

The reaction primarily targets exposed lysine residues on the antibody structure, allowing the fluorescent FITC molecule to become covalently attached while preserving the antibody's antigen recognition capability. This chemistry enables researchers to visualize and track antibody binding to target antigens in various applications .

What are the optimal reaction conditions for FITC conjugation to ENTR1 antibodies?

Research on FITC conjugation to antibodies indicates that optimal conditions for producing well-labeled conjugates include:

Specifically for ENTR1 antibodies, maintaining these conditions will produce conjugates with optimal fluorescein-to-protein (F/P) ratios . Research has demonstrated that a maximal molecular F/P ratio is achieved rapidly when reaction temperature, pH, and protein concentration are all optimized simultaneously .

How can I determine and optimize the fluorescein-to-protein (F/P) ratio for ENTR1 antibody conjugation?

The fluorescein-to-protein (F/P) ratio is critical for optimal performance of FITC-conjugated antibodies. For ENTR1 antibodies, as with other IgG antibodies, determining the F/P ratio requires spectrophotometric analysis:

• Measure the absorbance of the conjugate at 280 nm (A₂₈₀) and 495 nm (A₄₉₅)

• Calculate the F/P ratio using the formula:
  F/P = (A₄₉₅ × MW of antibody) / [(A₂₈₀ - 0.35 × A₄₉₅) × MW of FITC × ε₄₉₅]
  Where: ε₄₉₅ is the molar extinction coefficient of FITC (approximately 68,000 M⁻¹cm⁻¹)

Optimal F/P ratios typically range between 2.0-4.0 for most applications. Ratios below 2.0 may result in insufficient signal, while ratios above 6.0 can lead to quenching effects and potential interference with antibody binding .

For ENTR1 antibodies specifically, optimizing the conjugation conditions as described in section 1.3 will help achieve ideal F/P ratios. Research indicates that gradient DEAE Sephadex chromatography is an effective method for separating optimally labeled antibodies from under- and over-labeled proteins, allowing isolation of conjugates with the desired F/P ratio .

What purification methods are most effective for FITC-conjugated ENTR1 antibodies?

Several purification methods have been validated for FITC-conjugated antibodies, with different approaches offering specific advantages:

For ENTR1 antibodies, gradient DEAE Sephadex chromatography has been shown to be particularly effective for separating optimally labeled antibodies from under- and over-labeled proteins . This approach allows for isolation of conjugates with consistent F/P ratios, which is essential for reproducible experiments investigating ENTR1's role in endosomal trafficking and receptor regulation .

The purification step is critical, as research has demonstrated that antibody purification before conjugation significantly impacts labeling efficiency and specificity. Any contaminating proteins present will also be labeled, potentially compromising experimental results .

How should I validate the specificity and functionality of FITC-conjugated ENTR1 antibodies?

Validating FITC-conjugated ENTR1 antibodies requires multiple approaches to ensure both specificity for ENTR1 and retention of functional binding capacity after conjugation:

• Western Blot Validation:

  • Compare binding patterns of conjugated versus unconjugated antibodies

  • Verify detection of ENTR1 at expected molecular weight (~80 kDa)

  • Include known positive controls (e.g., cells with confirmed ENTR1 expression)

  • Include negative controls (e.g., ENTR1 knockout cells generated using CRISPR/Cas9)

• Immunofluorescence Validation:

  • Confirm localization pattern matches known ENTR1 distribution (endosomal punctae)

  • Co-staining with established endosomal markers (EEA1) should show co-localization

  • ENTR1 knockout or knockdown cells should show significantly reduced signal

• Flow Cytometry Validation:

  • Titrate antibody concentration (typically ≤0.5 μg mAb/million cells)

  • Include appropriate isotype controls (e.g., FITC-conjugated IgG of same isotype)

  • Pre-block with unconjugated antibody to demonstrate staining specificity

• Functional Validation:

  • Verify the antibody can detect changes in ENTR1 levels following siRNA knockdown

  • Confirm ability to detect ENTR1 in co-localization studies with cargo proteins (e.g., Fas receptor after stimulation)

Research has demonstrated that ENTR1 co-localizes with Fas in early endosomes following stimulation, providing a functional readout for validating antibody performance in trafficking studies .

How can FITC-conjugated ENTR1 antibodies be optimized for colocalization studies with endosomal markers?

Colocalization studies represent a powerful application for FITC-conjugated ENTR1 antibodies, particularly for investigating endosomal trafficking mechanisms. Optimizing these experiments requires several methodological considerations:

• Fixation and Permeabilization Protocol:

  • For ENTR1 studies, paraformaldehyde fixation (4%) followed by saponin permeabilization (0.1-0.5%) preserves endosomal structures while allowing antibody access

  • Avoid methanol fixation which can extract lipids and disrupt endosomal membranes

  • Short fixation times (10-15 minutes) minimize autofluorescence while maintaining structure

• Marker Selection for Multicolor Imaging:

  • Pair FITC-conjugated ENTR1 antibodies with spectrally distinct markers:

    • Early endosomes: Anti-EEA1 (conjugated to rhodamine or Alexa Fluor 555)

    • Late endosomes/lysosomes: Anti-LAMP1 (conjugated to Alexa Fluor 647)

    • Recycling endosomes: Anti-Rab11 (conjugated to Alexa Fluor 594)

  • Research has demonstrated significant ENTR1 colocalization with EEA1 but reduced colocalization with LAMP1 after receptor stimulation

• Image Acquisition Parameters:

  • Use sequential scanning to prevent bleed-through between channels

  • Match pinhole sizes across all channels for equivalent optical sections

  • Implement Nyquist sampling criteria to ensure sufficient resolution for colocalization analysis

• Quantitative Analysis Approaches:

  • Calculate Pearson's correlation coefficient and Mander's overlap coefficient

  • Perform object-based colocalization analysis for punctate endosomal structures

  • Analyze colocalization changes over time following receptor stimulation

Research has shown that ENTR1 colocalizes with Fas in punctate structures as early as five minutes after stimulation, consistent with endosomal localization . When ENTR1 is depleted, Fas shows increased colocalization with EEA1 (early endosomes) and decreased colocalization with LAMP1 (lysosomes), supporting ENTR1's role in endosomal sorting .

What are the most effective approaches for studying ENTR1-mediated receptor trafficking using FITC-conjugated antibodies?

Investigating ENTR1's role in receptor trafficking requires specialized experimental approaches that can be enhanced using FITC-conjugated antibodies:

• Pulse-Chase Receptor Trafficking Assays:

  • Surface-label receptors (e.g., Fas) with primary antibody

  • Stimulate internalization (e.g., with agonistic antibody CH11)

  • Fix cells at various timepoints and detect ENTR1 using FITC-conjugated antibodies

  • Quantify colocalization over time to track trafficking progression

• Enlarged Endosome Technique:

  • Transfect cells with constitutively active GFP-Rab5 to generate enlarged endosomes

  • This approach facilitates visualization of cargo in the limiting membrane versus lumen

  • Use FITC-conjugated ENTR1 antibodies to determine ENTR1 localization relative to cargo

  • Research has shown that in ENTR1-depleted cells, receptors like Fas predominantly localize to the limiting membrane rather than the lumen of enlarged endosomes

• Receptor Degradation Kinetics:

  • Stimulate receptor internalization and measure total receptor levels over time

  • Compare degradation rates in control versus ENTR1-depleted cells

  • Use FITC-conjugated ENTR1 antibodies for colocalization studies at each timepoint

  • Research indicates that ENTR1 depletion impairs transit from early endosomes to lysosomes

• Multivesicular Body Formation Analysis:

  • Examine ESCRT-dependent sorting into intraluminal vesicles

  • Analyze receptor distribution between limiting membrane and intraluminal vesicles

  • Studies demonstrate that in ENTR1-depleted cells, receptors accumulate at the limiting membrane of endosomes rather than sorting into intraluminal vesicles

These approaches have revealed that ENTR1 depletion does not affect endocytosis rates (as measured by surface Fas levels after stimulation) but significantly impairs post-endocytic sorting to lysosomes .

What strategies can minimize background and optimize signal-to-noise ratio when using FITC-conjugated ENTR1 antibodies?

Optimizing signal-to-noise ratio is critical for accurate interpretation of experiments using FITC-conjugated ENTR1 antibodies:

• Antibody Titration and Validation:

  • Perform careful titration experiments (≤0.5 μg mAb/million cells is recommended)

  • Include blocking controls by pre-incubating with unconjugated antibody

  • Use appropriate isotype controls at matched concentrations

• Reducing Autofluorescence Sources:

  • Minimize fixation time (10-15 minutes is typically sufficient)

  • Include quenching steps (e.g., 50mM NH₄Cl for 10 minutes after fixation)

  • For tissues or cells with high autofluorescence, consider:

    • Sudan Black B treatment (0.1-0.3% in 70% ethanol)

    • Copper sulfate treatment (10mM CuSO₄ in 50mM ammonium acetate)

• Optimizing F/P Ratio:

  • For FITC-conjugated antibodies, optimal F/P ratios (2-4) maximize signal while minimizing quenching

  • Purify conjugates to remove both under-labeled and over-labeled fractions

  • Gradient DEAE Sephadex chromatography effectively separates antibodies with different F/P ratios

• Image Acquisition Optimization:

  • Implement spectral unmixing for accurate separation of FITC signal from autofluorescence

  • Use narrow bandpass filters to isolate FITC emission (515-545nm)

  • Apply appropriate background subtraction methods during image analysis

• Reducing Non-Specific Binding:

  • Include thorough blocking steps (3-5% BSA, normal serum, or commercial blockers)

  • Add 0.1-0.3% Triton X-100 or 0.05-0.2% saponin to antibody diluent

  • Include 5-10% normal serum matching secondary antibody host species

Research demonstrates that proper antibody purification before conjugation significantly impacts specificity, as any contaminating proteins will also be labeled with FITC .

How should I quantify and interpret changes in ENTR1 localization in endosomal trafficking studies?

Quantitative analysis of ENTR1 localization requires rigorous methodological approaches:

• Colocalization Analysis Methods:

  • Calculate Pearson's correlation coefficient between ENTR1 and endosomal markers

  • Determine Mander's overlap coefficients (M1 and M2) to quantify fractional overlap

  • Apply intensity correlation analysis to assess dependent staining patterns

  • Research has shown significant changes in ENTR1 colocalization with cargo proteins (e.g., Fas) after receptor stimulation

• Endosomal Distribution Analysis:

  • Classify endosomal compartments using established markers:

    • Early endosomes (EEA1-positive)

    • Late endosomes/lysosomes (LAMP1-positive)

    • Recycling endosomes (Rab11-positive)

  • Calculate percentage of ENTR1 in each compartment under different conditions

  • Research demonstrates that ENTR1 primarily localizes to early endosomes

• Temporal Analysis of Trafficking:

  • Track changes in colocalization coefficients over time after receptor stimulation

  • Plot colocalization kinetics to identify rate-limiting steps in trafficking

  • Generate compartment transition matrices to model trafficking dynamics

• Statistical Approaches for Comparative Analysis:

  • Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple)

  • Use non-parametric alternatives for non-normally distributed data

  • Implement mixed-effects models for time-course experiments with multiple variables

Research has shown that when analyzing ENTR1-dependent trafficking, calculating the relative distribution between early endosomes (EEA1-positive) and lysosomes (LAMP1-positive) provides valuable insights. After 30 minutes of Fas activation, ENTR1-depleted cells showed significantly increased colocalization with EEA1 and decreased colocalization with LAMP1 compared to control cells .

What controls are essential when using FITC-conjugated antibodies for ENTR1 research?

Implementing appropriate controls is critical for generating reliable and interpretable data:

Research has demonstrated that these controls are essential for accurate interpretation. For example, experiments showed that increased Fas surface expression could be returned to normal levels by transfecting siRNA-resistant ENTR1 expression constructs, confirming the specificity of the observed phenotype .

How can I distinguish between direct and indirect effects of ENTR1 on receptor trafficking pathways?

Differentiating direct from indirect effects of ENTR1 on receptor trafficking requires sophisticated experimental approaches:

• Temporal Resolution Studies:

  • Track the sequence of events following receptor stimulation

  • Research shows ENTR1 colocalizes with Fas in punctate structures as early as five minutes after stimulation, suggesting direct involvement

  • Compare timing of ENTR1 recruitment with other trafficking regulators

• Domain Mapping and Mutational Analysis:

  • Generate ENTR1 constructs with mutations in functional domains

  • Analyze which domains are required for endosomal localization versus receptor interaction

  • Compare trafficking defects with different mutants to identify specific functional requirements

• Biochemical Interaction Studies:

  • Perform co-immunoprecipitation under different conditions (unstimulated vs. stimulated)

  • Analyze timing of complex formation relative to trafficking events

  • Research indicates that while ENTR1 appears constitutively localized to endosomes, other interactors like PTPN13 are only recruited to endosomes after Fas stimulation

• Cargo-Specific Effects Analysis:

  • Compare ENTR1's effect on multiple different receptor cargoes

  • Determine whether effects are universal or cargo-specific

  • Research demonstrates ENTR1 depletion affects Fas trafficking in both HeLa and HCT116 cell lines, suggesting a conserved mechanism

• Multivesicular Body Formation Analysis:

  • Examine the effect of ENTR1 depletion on cargo sorting into intraluminal vesicles

  • Research shows that in ENTR1-depleted cells, Fas localizes predominantly to the limiting membrane of enlarged endosomes rather than the lumen, suggesting a direct role in ESCRT-mediated sorting

These approaches have revealed that while ENTR1 depletion does not affect endocytosis kinetics, it significantly impairs the transition from early endosomes to lysosomes, indicating a specific role in post-endocytic sorting rather than internalization .

How do FITC-conjugated ENTR1 antibodies compare with other fluorophore conjugates for advanced microscopy applications?

Different fluorophores offer distinct advantages for various applications in ENTR1 research:

For ENTR1 studies specifically:

• FITC-conjugated antibodies are well-suited for standard colocalization analyses with endosomal markers using confocal microscopy

• Flow cytometry applications benefit from FITC's strong signal and compatibility with standard filter sets

• For advanced live-cell imaging tracking ENTR1-dependent trafficking, more photostable alternatives like Alexa Fluor 488 may be preferable

• Studies examining fine endosomal subdomains may benefit from super-resolution approaches using photoconvertible fluorophores

Research indicates that FITC-conjugated antibodies can effectively visualize ENTR1 in fixed cells for colocalization studies with endosomal markers. The optimal fluorophore choice depends on the specific application, imaging modality, and experimental duration .

What are emerging applications for FITC-conjugated ENTR1 antibodies in multi-parameter flow cytometry?

Multiparameter flow cytometry offers powerful approaches for analyzing ENTR1's role in cellular processes:

• Receptor Trafficking Analysis:

  • Combine surface and intracellular staining to track receptor internalization

  • Design panels incorporating:

    • FITC-conjugated ENTR1 antibodies for intracellular detection

    • APC-conjugated receptor antibodies (e.g., anti-Fas) for surface expression

    • PE-conjugated endosomal markers for compartment identification

  • Quantify changes in surface receptor levels upon ENTR1 depletion or overexpression

• Cell-Cycle Dependent Trafficking:

  • Integrate DNA content staining (e.g., DAPI or Hoechst) with ENTR1 and receptor analysis

  • Investigate whether ENTR1-dependent trafficking varies across cell cycle phases

  • Correlate ENTR1 expression levels with cell cycle progression

• Phospho-Flow Applications:

  • Combine phospho-specific antibodies with ENTR1 detection

  • Analyze how signaling cascades correlate with ENTR1 expression

  • Investigate feedback between receptor signaling and trafficking

• Apoptosis and Cell Fate Studies:

  • Develop panels incorporating:

    • FITC-conjugated ENTR1 antibodies

    • Annexin V conjugates (e.g., APC-Annexin V)

    • Mitochondrial potential dyes (e.g., TMRE)

  • Research has shown ENTR1's importance in regulating Fas surface levels, which directly impacts apoptotic signaling

• Panel Design Considerations:

  • Apply compensation controls for each fluorophore

  • Include FMO (Fluorescence Minus One) controls

  • Implement specialized fixation protocols to preserve both surface and intracellular epitopes

  • Use appropriate isotype controls at matched concentrations (e.g., FITC-MOPC-21)

For intracellular flow cytometry applications, research recommends careful titration of antibodies (≤0.5 μg mAb/million cells) and application of specialized fixation/permeabilization protocols to maintain both antigen detection and cellular integrity .

What novel methodological approaches can enhance the specificity and sensitivity of ENTR1 trafficking studies?

Advanced methodological approaches continue to evolve for investigating ENTR1's role in endosomal trafficking:

• Proximity Ligation Assay (PLA):

  • Detect direct protein-protein interactions between ENTR1 and cargo or trafficking machinery

  • Provides single-molecule resolution of interactions in situ

  • Can detect transient interactions that may be missed by colocalization studies

  • Particularly valuable for identifying direct ENTR1 binding partners during trafficking

• Super-Resolution Microscopy Applications:

  • STORM/PALM imaging reveals nanoscale organization of ENTR1 within endosomal subdomains

  • Structured illumination microscopy (SIM) improves resolution while maintaining multicolor capability

  • Expansion microscopy physically enlarges samples for enhanced resolution with standard confocal microscopy

  • These approaches can reveal ENTR1 distribution within endosomal subdomains not resolvable by conventional microscopy

• Correlative Light and Electron Microscopy (CLEM):

  • Combine fluorescence localization of ENTR1 with ultrastructural analysis

  • Particularly valuable for analyzing ENTR1's role in multivesicular body formation

  • Research suggests ENTR1 influences cargo sorting into intraluminal vesicles, which can be directly visualized using CLEM

• CRISPR-Based Approaches:

  • Generate endogenous fluorescent protein fusions using CRISPR knock-in

  • Create ENTR1 knockout cell lines for definitive loss-of-function studies

  • Implement CRISPR interference (CRISPRi) for tunable and reversible ENTR1 depletion

  • Research has successfully utilized CRISPR/Cas9 to generate ENTR1 knockout HCT116 cells for trafficking studies

• Live-Cell Trafficking Analysis:

  • Implement photoactivatable or photoconvertible fluorescent protein fusions

  • Enable pulse-chase studies of specific endosomal populations

  • Allow direct visualization of cargo sorting and trafficking in real-time

  • Particularly valuable for determining precise kinetics of ENTR1-dependent trafficking steps

These advanced approaches can provide deeper insights into ENTR1's molecular mechanism. Research using the enlarged endosome technique (with constitutively active GFP-Rab5) revealed that ENTR1 depletion specifically impairs cargo sorting from the limiting membrane into the lumen of multivesicular bodies, suggesting a direct role in ESCRT-mediated sorting mechanisms .