ENHO Antibody, FITC conjugated

What is ENHO protein and why is it significant in metabolic research?

ENHO (Energy Homeostasis Associated) is a protein involved in the regulation of glucose homeostasis and lipid metabolism. As a key regulator in metabolic pathways, ENHO (also known as adropin) has gained significant interest in research related to cardiovascular health, metabolic diseases, and energy balance regulation. The protein is encoded by the ENHO gene located on chromosome 9 in humans, with the UniProt ID Q6UWT2. Research interest in ENHO stems from its potential role in metabolic disorders including obesity, diabetes, and cardiovascular disease, making it an important target for immunological detection and quantification in various experimental systems .

What is antibody conjugation and how does FITC labeling work?

Antibody conjugation is the process of chemically linking an antibody to another molecule, such as a fluorescent dye, enzyme, biotin, or nanoparticle. This process enhances the antibody's detection capabilities by enabling visualization or measurement in various assays. Specifically, FITC conjugation involves the chemical attachment of the fluorescent dye fluorescein isothiocyanate to an antibody molecule, typically through covalent bonding to primary amine groups on the antibody. This conjugation occurs most commonly at lysine residues or the N-terminus of the antibody's heavy and light chains .

The conjugation chemistry typically utilizes the isothiocyanate group of FITC, which reacts with primary amines on the antibody under slightly alkaline conditions (pH 8.0-9.5) to form stable thiourea bonds. The reaction must be carefully controlled to ensure sufficient labeling without compromising the antibody's antigen-binding capacity. Modern conjugation kits like Lightning-Link® systems have simplified this process, allowing researchers to conjugate antibodies in just three simple steps while maintaining antibody functionality .

What are the key specifications of commercially available ENHO Antibody, FITC conjugated products?

Commercial ENHO Antibody, FITC conjugated products typically have the following specifications:

This information helps researchers select the appropriate antibody for their specific experimental needs and understand the product's properties for optimal use in laboratory settings .

What are the principal applications for ENHO Antibody, FITC conjugated in research?

ENHO Antibody, FITC conjugated serves multiple research applications, particularly in studies investigating energy metabolism, glucose homeostasis, and lipid regulation. The principal applications include:

• Immunofluorescence microscopy: For visualizing the subcellular localization of ENHO protein in tissue sections or cultured cells. The direct FITC conjugation eliminates the need for secondary antibody incubation steps, reducing background and cross-reactivity issues while simplifying the experimental workflow .

• Flow cytometry: For quantitative analysis of ENHO expression in cell populations. The FITC fluorophore is excited at 488 nm and emits green fluorescence at approximately 520 nm, making it compatible with standard flow cytometry lasers and filter sets. This application is particularly valuable for studying ENHO expression in different cell types or under various metabolic conditions .

• Confocal microscopy: For high-resolution imaging of ENHO protein distribution in cells and tissues, enabling detailed analysis of its co-localization with other cellular structures or proteins when combined with additional fluorophore-labeled antibodies targeting other proteins of interest .

• Multiplex immunostaining: FITC's spectral characteristics allow it to be combined with other fluorophores with minimal spectral overlap, enabling simultaneous detection of multiple targets in the same sample .

These applications make FITC-conjugated ENHO antibodies valuable tools in metabolism research, cardiovascular studies, and investigations of metabolic disorders .

What is the optimal protocol for immunofluorescence using ENHO Antibody, FITC conjugated?

A methodologically sound protocol for immunofluorescence using ENHO Antibody, FITC conjugated includes the following steps:

• Sample preparation:

  • Fix cells or tissue sections with 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • Block with 5% normal serum (from the same species as the secondary antibody would be if using an indirect method) in PBS with 0.1% Tween-20 for 1 hour at room temperature

• Antibody incubation:

  • Dilute the ENHO Antibody, FITC conjugated to the optimal working concentration (typically 1-10 μg/mL, but this should be empirically determined)

  • Apply to the sample and incubate in a humidified chamber for 1-2 hours at room temperature or overnight at 4°C

  • Protect from light throughout the incubation to prevent photobleaching of the FITC fluorophore

• Washing steps:

  • Wash 3-5 times with PBS containing 0.1% Tween-20, 5 minutes each

  • Perform all washes in the dark or under reduced lighting

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (4′,6-diamidino-2-phenylindole) at 0.1-1 μg/mL for 5-10 minutes

  • Mount using an anti-fade mounting medium to minimize photobleaching

  • Seal the edges of the coverslip with nail polish or commercial sealant

• Imaging considerations:

  • Use appropriate filter sets for FITC (excitation: 490 nm, emission: 525 nm)

  • Include an unstained control and an isotype control to assess autofluorescence and non-specific binding

  • Capture images promptly to minimize photobleaching or store slides at 4°C in the dark

This protocol should be optimized for each specific experimental system and research question.

How can researchers address weak signal issues when using ENHO Antibody, FITC conjugated?

When encountering weak signal intensity with ENHO Antibody, FITC conjugated, researchers should systematically troubleshoot the following potential causes:

• Antibody concentration: Insufficient antibody concentration is a common cause of weak signals. Perform a titration experiment using different antibody concentrations (0.5-20 μg/mL) to determine the optimal working concentration for your specific sample type. Unlike enzyme-conjugated antibodies which offer signal amplification, fluorophore-conjugated antibodies provide direct visualization, requiring careful optimization of concentration .

• Target protein expression levels: ENHO may be expressed at low levels in certain tissues or under specific conditions. Consider using positive control samples known to express ENHO abundantly (such as liver tissue) to validate antibody performance. Alternative detection methods with higher sensitivity may be necessary for low-abundance targets .

• Sample preparation issues:

  • Inadequate fixation or over-fixation can mask epitopes

  • Insufficient permeabilization can prevent antibody access to intracellular targets

  • Suboptimal antigen retrieval (for FFPE tissues) can prevent antibody binding

  • Test different fixation methods and permeabilization conditions to optimize epitope accessibility

• Fluorophore deterioration:

  • FITC is relatively susceptible to photobleaching compared to more modern fluorophores

  • Check the antibody's manufacturing date and storage conditions

  • Consider using an alternative fluorophore with greater photostability (such as Alexa Fluor 488) if photobleaching is a significant issue

  • Always store the conjugate at recommended temperatures (-20°C to -80°C) and protect from light exposure during all steps

• Signal enhancement strategies:

  • Increase the exposure time during image acquisition, balancing against background increase

  • Use a more sensitive detection system (e.g., confocal microscopy, PMT-based detectors)

  • Consider signal amplification using anti-FITC antibodies or tyramide signal amplification if compatible with your experimental design

By systematically addressing these factors, researchers can optimize signal detection for ENHO using FITC-conjugated antibodies.

What strategies reduce background fluorescence when using FITC-conjugated antibodies?

High background fluorescence is a common challenge when working with FITC-conjugated antibodies. Researchers can implement the following methodological approaches to reduce background and improve signal-to-noise ratio:

• Optimize blocking conditions:

  • Use 5-10% normal serum from the same species as the host of the primary antibody

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to blocking buffers to reduce non-specific binding

  • Consider adding 1-5% BSA to further reduce non-specific interactions

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C for challenging samples

• Reduce autofluorescence:

  • For tissues with high autofluorescence (particularly formalin-fixed tissues), treat with 0.1-1% sodium borohydride in PBS for 10 minutes before blocking

  • For samples with lipofuscin (common in aged tissues), incubate with 0.1-0.3% Sudan Black B in 70% ethanol for 20 minutes

  • Include unstained controls to assess the level of tissue autofluorescence

• Improve washing steps:

  • Increase the number of washes (5-6 washes instead of 3)

  • Extend washing times to 10-15 minutes per wash

  • Include 0.05-0.1% Tween-20 in wash buffers

  • Use gentle agitation during washing to remove unbound antibody effectively

• Address antibody quality issues:

  • Centrifuge antibody solutions (10,000 × g for 5 minutes) before use to remove aggregates

  • Filter antibody dilutions through a 0.22 μm filter if aggregation is a persistent problem

  • Validate antibody specificity using appropriate controls (western blot, competitive blocking with immunizing peptide)

• Optimize imaging parameters:

  • Adjust the dynamic range during image acquisition to maximize signal while minimizing background

  • Use narrower bandpass filters to reduce spectral bleed-through

  • Employ computational background subtraction during image processing when appropriate

By implementing these methods systematically, researchers can significantly improve the signal-to-noise ratio when using ENHO Antibody, FITC conjugated in their experiments.

How can ENHO Antibody, FITC conjugated be incorporated into multiplex immunofluorescence assays?

Multiplex immunofluorescence allows for simultaneous detection of multiple targets in a single sample, providing valuable co-expression and co-localization data. To effectively incorporate ENHO Antibody, FITC conjugated into multiplex panels, researchers should follow these methodological guidelines:

• Spectral compatibility planning:

  • FITC has excitation/emission maxima at approximately 490/525 nm (green fluorescence)

  • Select additional fluorophores with minimal spectral overlap, such as:

    • Cy3 or PE (excitation: ~550 nm, emission: ~570 nm) for red fluorescence

    • Cy5, APC, or Alexa Fluor 647 (excitation: ~650 nm, emission: ~670 nm) for far-red fluorescence

    • DAPI (excitation: ~350 nm, emission: ~450 nm) for nuclear counterstaining

• Panel design considerations:

  • Reserve FITC for medium-to-high abundance targets like ENHO when expression levels are strong

  • For lower abundance targets, consider brighter fluorophores like PE or APC

  • Account for potential cross-reactivity between antibodies by selecting primaries raised in different host species when using indirect detection methods alongside your direct FITC conjugate

• Sequential staining approach:

  • When combining directly conjugated antibodies with indirect detection (primary + secondary):

    • First apply all unconjugated primary antibodies

    • Then apply fluorophore-conjugated secondary antibodies

    • Block any remaining active sites on secondary antibodies

    • Finally apply directly conjugated antibodies like ENHO-FITC

  • This prevents secondary antibodies from recognizing the FITC-conjugated primary

• Controls for multiplex panels:

  • Single-color controls: Stain separate samples with each individual antibody to confirm proper localization and assess bleed-through

  • Fluorescence-minus-one (FMO) controls: Include panels with each fluorophore omitted to identify spectral overlap issues

  • Isotype controls: Include appropriate isotype controls for each antibody to assess non-specific binding

• Image acquisition and analysis considerations:

  • Use sequential scanning (for confocal microscopy) to minimize spectral crosstalk

  • Employ spectral unmixing algorithms for fluorophores with partial overlap

  • Consider using ENHO Antibody conjugated to alternative fluorophores (e.g., Cy3 or Alexa Fluor 647) if spectral conflicts emerge with other targets in your panel

By carefully designing multiplex panels and following these methodological guidelines, researchers can effectively incorporate ENHO Antibody, FITC conjugated into complex immunofluorescence studies examining multiple markers simultaneously.

What approaches can quantify ENHO expression levels using FITC-conjugated antibodies?

Quantifying ENHO expression using FITC-conjugated antibodies requires systematic approaches to ensure accuracy and reproducibility. Researchers can employ the following quantitative methods:

• Flow cytometry-based quantification:

  • Prepare single-cell suspensions from tissues or cultured cells

  • Stain with ENHO Antibody, FITC conjugated using optimized concentrations

  • Include calibration beads with known fluorescence intensities to convert arbitrary fluorescence units to molecules of equivalent soluble fluorochrome (MESF)

  • Analyze mean fluorescence intensity (MFI) or median fluorescence intensity of positive populations

  • Present data as fold-change relative to controls or as absolute MESF values

  • Incorporate appropriate controls including isotype-FITC control, unstained cells, and positive/negative control samples

• Quantitative immunofluorescence microscopy:

  • Capture images under identical acquisition settings for all experimental conditions

  • Include internal reference standards in each experiment (such as fluorescent microspheres)

  • Apply thresholding to segment positive areas from background

  • Quantify parameters such as:

    • Mean fluorescence intensity per cell

    • Integrated density (the product of area and mean gray value)

    • Percent positive area in tissue sections

  • Normalize to cell number using nuclear counterstains or to tissue area for comparative analyses

• Digital image analysis workflows:

  • Use software platforms like ImageJ/FIJI, CellProfiler, or QuPath for automated quantification

  • Develop analysis pipelines that include:

    • Nuclear segmentation (using DAPI)

    • Cell boundary identification

    • Background subtraction

    • Intensity measurement within defined cellular compartments

  • Apply batch processing for consistent analysis across multiple images

  • Export quantitative data for statistical analysis in programs like R or GraphPad Prism

• Statistical considerations for quantitative analyses:

  • Account for non-normal distributions in fluorescence intensity data

  • Apply appropriate transformations (log, square root) before parametric statistical tests

  • Consider cell-by-cell analysis rather than population averages when heterogeneity is expected

  • Report both technical and biological replicates with appropriate measures of central tendency and dispersion

By implementing these quantitative approaches, researchers can obtain reliable measurements of ENHO expression levels using FITC-conjugated antibodies across various experimental systems and conditions.

How does FITC compare to other fluorophore conjugates for ENHO detection?

When selecting fluorophore conjugates for ENHO detection, researchers should consider the relative advantages and limitations of FITC compared to alternative fluorophores. The following comparative analysis provides guidance for informed decision-making:

For ENHO detection specifically:

• For routine applications (basic immunofluorescence, flow cytometry):

  • FITC conjugates provide adequate performance with standard equipment

  • Consider when budget constraints exist or when comparing to historical FITC-based data

• For challenging samples (low ENHO expression, high autofluorescence tissues):

  • Alexa Fluor 488 offers superior performance with similar spectral properties to FITC

  • R-PE provides significantly higher brightness for detecting low abundance ENHO

• For advanced imaging applications (confocal, super-resolution):

  • Alexa Fluor 488 or iFluor 488 conjugates offer superior photostability

  • Quantum dot conjugates may be considered for long-term imaging or when extreme photostability is required

• For multiplexing considerations:

  • When designing multiplex panels, the spectral properties of each fluorophore must be considered holistically

  • FITC occupies the green channel, leaving red and far-red channels available for other targets

  • For complex multiplexing, spectral unmixing with narrower bandwidth fluorophores may be advantageous

This comparative analysis enables researchers to select the optimal fluorophore conjugate for their specific ENHO detection needs based on experimental requirements, equipment availability, and research goals.

When should researchers consider alternative detection methods to fluorophore-conjugated antibodies for ENHO studies?

While FITC-conjugated antibodies are valuable tools for ENHO detection, alternative methodologies may be more appropriate in certain research contexts. Researchers should consider the following situations and alternative approaches:

• For quantitative protein expression analysis:

  • Enzyme-linked immunosorbent assay (ELISA): When precise quantification of ENHO levels in solution is required, ELISA using HRP or AP conjugates offers superior quantification compared to fluorescence-based methods

  • Western blotting with enzyme conjugates: For determining protein size, post-translational modifications, or when sample autofluorescence is problematic

  • Methodology: Use unconjugated anti-ENHO primary antibody followed by enzyme-conjugated secondary antibody (HRP or AP) for enhanced sensitivity through signal amplification

• For challenging tissue samples:

  • Immunohistochemistry with chromogenic detection: When working with tissues having high autofluorescence (e.g., liver, kidney) or with archived FFPE samples

  • Metal-conjugated antibodies for mass cytometry (CyTOF): For high-dimensional analysis without fluorescence spectral overlap limitations

  • Methodology: Use biotinylated anti-ENHO antibody followed by streptavidin-HRP and DAB substrate for chromogenic detection in difficult tissues

• For high sensitivity requirements:

  • Tyramide signal amplification (TSA): When detecting very low levels of ENHO expression

  • Biotin-streptavidin amplification systems: For enhanced signal through multiple binding sites

  • Methodology: Use unconjugated primary anti-ENHO antibody, followed by biotinylated secondary antibody, then streptavidin-HRP and tyramide-fluorophore for exponential signal amplification

• For live cell applications:

  • Genetic reporters: When studying ENHO dynamics in living cells

  • Antibody fragments or nanobodies: For improved tissue penetration and reduced immunogenicity

  • Methodology: Generate ENHO-GFP fusion constructs through genetic engineering or use anti-ENHO Fab fragments labeled with pH-stable fluorophores

• For spatial context in tissue architecture:

  • Multiplex immunohistochemistry with sequential chromogenic detection: When spatial relationships between ENHO and multiple markers are critical

  • RNA in situ hybridization: When antibody specificity is a concern or to correlate protein with mRNA expression

  • Methodology: Use RNAscope or similar technology to detect ENHO mRNA in conjunction with protein detection

The decision to use alternative detection methods should be guided by the specific research question, sample characteristics, required sensitivity, and available instrumentation. Each method offers distinct advantages in particular experimental contexts, and researchers may benefit from employing multiple complementary approaches to validate and extend their findings on ENHO biology.

How should researchers validate the specificity of ENHO Antibody, FITC conjugated?

Validating antibody specificity is crucial for ensuring reliable and reproducible research outcomes. For ENHO Antibody, FITC conjugated, researchers should implement the following methodological validation strategies:

• Positive and negative control samples:

  • Use tissues or cell lines with well-documented ENHO expression as positive controls

  • Include samples known to lack ENHO expression as negative controls

  • Compare staining patterns between controls to confirm expected differential expression patterns

• Genetic validation approaches:

  • Compare staining in wild-type versus ENHO knockout models (if available)

  • Use ENHO-overexpressing systems as positive controls

  • Employ siRNA or shRNA knockdown of ENHO to demonstrate reduced staining intensity

  • These genetic approaches represent the gold standard for antibody validation

• Peptide competition assays:

  • Pre-incubate the ENHO Antibody, FITC conjugated with excess immunizing peptide (the peptide sequence from Human Adropin protein AA 37-54 used to generate the antibody)

  • Compare staining with and without peptide pre-absorption

  • Specific staining should be significantly reduced or eliminated after peptide competition

• Orthogonal method validation:

  • Confirm ENHO expression using independent methods such as:

    • Western blotting with different anti-ENHO antibodies

    • Mass spectrometry-based protein identification

    • RNA expression analysis (RT-qPCR or RNA-seq)

  • Correlation between methods strengthens confidence in antibody specificity

• Multi-antibody concordance:

  • Compare staining patterns obtained with multiple antibodies against different ENHO epitopes

  • Similar patterns across different antibodies increase confidence in specificity

  • Document discrepancies between antibodies for transparent reporting

• Publication and reporting standards:

  • Document all validation steps performed

  • Report the specific catalog number, lot number, and dilution used

  • Include representative images of positive and negative controls

  • Disclose any limitations in specificity or cross-reactivity observed

  • Follow reporting guidelines such as those proposed by the International Working Group for Antibody Validation (IWGAV)

By implementing these validation strategies, researchers can establish confidence in the specificity of their ENHO Antibody, FITC conjugated and produce more reliable and reproducible research findings.

What are the critical considerations for interpreting co-localization data with ENHO Antibody, FITC conjugated?

Co-localization analysis using ENHO Antibody, FITC conjugated requires careful methodological considerations to ensure accurate data interpretation. Researchers should address the following critical aspects: