TKFC Antibody, FITC conjugated

What is TKFC protein and what are its biological functions?

TKFC (Triokinase/FMN cyclase) is a bifunctional enzyme with two distinct catalytic activities. It catalyzes the phosphorylation of dihydroxyacetone and glyceraldehyde, and also mediates the splitting of ribonucleoside diphosphate-X compounds, with FAD being its preferred substrate . Additionally, TKFC plays a regulatory role in cellular antiviral responses by repressing IFIH1-mediated pathways . The protein is also known by several alias names including ATP-dependent dihydroxyacetone kinase, FAD-AMP lyase (cyclizing), FMN cyclase, DHA kinase, glycerone kinase, triokinase, and triose kinase . In human systems, TKFC is identified by the UniProt Primary accession number Q3LXA3, with additional secondary accession numbers including Q2L9C1, Q53EQ9, Q9BVA7, and Q9H895 .

What are the structural characteristics of commercially available TKFC Antibody, FITC conjugated?

The commercially available TKFC Antibody, FITC conjugated is a polyclonal antibody raised in rabbits using recombinant Human Triokinase/FMN cyclase protein (specifically amino acids 396-561) as the immunogen . The antibody has been purified using Protein G chromatography, resulting in a highly pure preparation (>95% purity) . This antibody demonstrates specific reactivity with human TKFC protein and is of the IgG isotype . The FITC conjugation provides fluorescence capabilities with excitation/emission wavelengths of 499/515 nm, making it compatible with the 488 nm laser line commonly used in flow cytometry and fluorescence microscopy applications .

What are the recommended storage conditions for maintaining TKFC Antibody, FITC conjugated stability?

Proper storage is critical for maintaining antibody functionality and fluorophore integrity. TKFC Antibody, FITC conjugated should be stored at -20°C (short-term) or -80°C (long-term) . The antibody is typically provided in a stabilizing buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . To maintain optimal activity:

• Aliquot the antibody upon receipt to avoid repeated freeze-thaw cycles

• Store aliquots at -20°C in the dark

• Protect from light exposure during storage and handling to prevent photobleaching of the FITC fluorophore

• Avoid repeated freeze-thaw cycles that can denature the antibody and reduce fluorescence intensity

For working solutions, temporary storage at 2-8°C for up to one month is possible under sterile conditions, though prolonged storage at this temperature is not recommended .

What are the basic principles of FITC conjugation to antibodies?

FITC (fluorescein isothiocyanate) conjugation to antibodies involves the covalent attachment of the fluorescent molecule to primary amine groups on the antibody, primarily at lysine residues. The conjugation reaction is pH-dependent and typically performed under alkaline conditions (pH 9.5) to ensure that lysine residues are deprotonated and reactive . The molecular ratio of fluorescein to protein (F/P ratio) is a critical parameter that determines the brightness and functionality of the conjugated antibody.

Key factors affecting conjugation efficiency include:

• Reaction time: Maximal labeling is typically achieved within 30-60 minutes at room temperature

• pH: Optimal conjugation occurs at pH 9.5

• Protein concentration: Higher initial protein concentrations (approximately 25 mg/ml) promote efficient labeling

• Antibody purity: Using highly purified IgG, such as that obtained by DEAE Sephadex chromatography, ensures more consistent conjugation results

After conjugation, separation of optimally labeled antibodies from under- and over-labeled proteins is typically achieved using gradient DEAE Sephadex chromatography .

What are the primary applications for TKFC Antibody, FITC conjugated?

TKFC Antibody, FITC conjugated can be utilized in multiple immunological applications, with particular emphasis on fluorescence-based techniques. The primary applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Both standard ELISA and fluorescence-based ELISA (FLISA) can be performed with recommended dilutions of 1:1000 for FLISA applications .

• Flow Cytometry: The antibody can be used for multicolor immunofluorescent staining to identify and enumerate TKFC-expressing cells within heterogeneous cell populations. For optimal staining, the antibody should be titrated (typically ≤0.5 μg antibody per million cells) .

• Immunofluorescence Microscopy: The FITC conjugate enables direct visualization of TKFC localization in fixed cells or tissue sections without the need for secondary antibody detection.

• Western Blot: Although less common for fluorescent conjugates, the antibody can be used for Western blot applications at dilutions of 1:100-500 .

When designing experiments, researchers should determine optimal concentrations for their specific application through titration, as antibody performance can vary depending on sample type, preparation method, and detection system .

What controls should be included when using TKFC Antibody, FITC conjugated?

Proper experimental controls are essential for ensuring reliable and interpretable results when using TKFC Antibody, FITC conjugated. The following controls should be considered:

• Isotype Control: An isotype-matched FITC-conjugated antibody (rabbit IgG-FITC) should be used at the same concentration as the TKFC antibody to assess the level of background staining and non-specific binding .

• Blocking Controls: To demonstrate staining specificity, consider:

  • Pre-blocking the conjugated antibody with recombinant TKFC protein before staining

  • Pre-blocking fixed/permeabilized cells with unlabeled TKFC antibody before staining with the FITC-conjugated version

• Negative Control Samples: Include samples known to be negative for TKFC expression to establish background fluorescence levels.

• Positive Control Samples: Include samples with confirmed TKFC expression to validate antibody performance.

• Fluorescence Minus One (FMO) Controls: In multicolor flow cytometry experiments, include controls where each fluorochrome is omitted one at a time to accurately set compensation parameters.

• Unstained Control: Include completely unstained samples to assess autofluorescence in your cell type or tissue.

These controls help differentiate between specific signal and background, enabling accurate data interpretation and troubleshooting of unexpected results .

How should sample preparation be optimized for TKFC Antibody, FITC conjugated in intracellular staining?

Optimization of sample preparation is crucial for successful intracellular staining with TKFC Antibody, FITC conjugated. The following methodological considerations should be addressed:

• Fixation Protocol:

  • Use paraformaldehyde (typically 2-4%) for 10-15 minutes at room temperature

  • Avoid methanol-based fixatives which can reduce FITC fluorescence

  • Wash cells thoroughly after fixation to remove residual paraformaldehyde

• Permeabilization:

  • Use saponin (0.1-0.5%) for reversible membrane permeabilization

  • Alternative permeabilization agents include Triton X-100 (0.1%) for stronger permeabilization

  • Maintain permeabilization agent in all wash buffers throughout the staining procedure

• Blocking:

  • Incubate samples with 5-10% normal serum (from the same species as secondary antibodies if used) to reduce non-specific binding

  • Include human FcR blocking reagent when working with human samples

• Antibody Titration:

  • Determine optimal antibody concentration through titration experiments

  • Start with recommended dilution and test 2-3 concentrations above and below

• Incubation Conditions:

  • Perform staining in the dark to protect FITC from photobleaching

  • Incubate at 4°C for 30-60 minutes for optimal staining

  • For difficult epitopes, consider extending incubation time or performing at room temperature

• Washing Steps:

  • Use sufficient washing steps (at least 3) with adequate buffer volume

  • Include permeabilization agent in wash buffer for intracellular targets

Following these methodological steps will help optimize the signal-to-noise ratio and increase the specificity of TKFC detection in your samples .

How does FITC photobleaching affect experiments with TKFC Antibody, FITC conjugated and what strategies can minimize this effect?

FITC is more susceptible to photobleaching than many other fluorophores, which can significantly impact experimental outcomes with TKFC Antibody, FITC conjugated. Photobleaching is the photochemical destruction of the fluorophore resulting from exposure to excitation light, leading to decreasing signal intensity over time.

The practical implications of photobleaching include:

• Reduced sensitivity in detecting low-abundance TKFC

• Inaccurate quantification in time-lapse experiments

• Misleading comparative analysis between samples imaged at different times

To minimize photobleaching effects, implement these methodological strategies:

• Anti-fade Reagents:

  • Include anti-fade reagents in mounting media or buffer solutions

  • Commercial products containing p-phenylenediamine or propyl gallate significantly extend FITC fluorescence lifetime

• Imaging Optimization:

  • Reduce exposure time and excitation light intensity to the minimum needed for detection

  • Use neutral density filters to attenuate excitation light

  • Close the field diaphragm to illuminate only the area being observed

• Sequential Acquisition Strategy:

  • When performing multicolor imaging, acquire FITC channel first

  • Plan the shortest possible imaging session for FITC visualization

• Oxygen Scavenger Systems:

  • Include enzymatic oxygen scavenging systems (e.g., glucose oxidase/catalase) in imaging buffers

  • Oxygen removal significantly reduces photobleaching rates of fluorescein derivatives

• Sample Preparation:

  • Maintain samples at 4°C during preparation to reduce thermal-induced fluorophore degradation

  • Protect samples from ambient light from the conjugation step through analysis

Quantitative assessment of photobleaching can be performed by measuring fluorescence intensity over sequential exposures, allowing researchers to calculate correction factors for quantitative applications when necessary.

What are the theoretical and practical considerations for determining optimal fluorescein/protein (F/P) ratio in FITC-conjugated antibodies?

The fluorescein/protein (F/P) ratio is a critical parameter that influences the performance of FITC-conjugated antibodies, including TKFC Antibody, FITC conjugated. This ratio represents the average number of fluorescein molecules attached to each antibody molecule and directly impacts brightness, specificity, and functionality.

Theoretical Considerations:

The optimal F/P ratio typically falls within a specific range:

• Too low (<2): Results in insufficient brightness for detection of low-abundance targets

• Optimal (2-8): Provides adequate brightness while maintaining antibody specificity and affinity

• Too high (>8): Can cause steric hindrance, increased hydrophobicity, self-quenching, and reduced antibody affinity

Methodological Approaches for F/P Ratio Determination:

• Spectrophotometric Method:

  • Measure absorbance at 280 nm (protein) and 495 nm (FITC)

  • Calculate F/P ratio using the formula:
    F/P = (A495 × C) / [(A280 - (A495 × 0.35)) × P]
    where C is the correction factor for FITC, and P is the protein extinction coefficient

• Chromatographic Separation:

  • Gradient DEAE Sephadex chromatography can separate antibody populations with different F/P ratios

  • Collect fractions and analyze for both protein content and fluorescence intensity

• Fluorescence Correlation Spectroscopy:

  • For more precise measurements, advanced techniques like FCS can determine the exact distribution of F/P ratios within an antibody preparation

Practical Optimization Guidelines:

To achieve optimal F/P ratios in laboratory settings:

• Control reaction conditions: pH 9.5, room temperature, 30-60 minutes, protein concentration of 25 mg/ml

• Use high-quality, pure IgG preparations for conjugation

• Test different FITC-to-protein molar ratios in parallel conjugation reactions

• Evaluate antibody performance across different F/P ratios in your specific application

• Consider that electrophoretically distinct IgG molecules demonstrate similar affinity for FITC

These considerations ensure that TKFC Antibody, FITC conjugated will provide optimal brightness while maintaining target specificity and binding affinity.

How can multiplexing experiments be designed with TKFC Antibody, FITC conjugated?

Multiplexing with TKFC Antibody, FITC conjugated enables simultaneous detection of multiple targets, providing contextual information about TKFC expression relative to other markers. Successful multiplexing requires careful experimental design to address spectral overlap and ensure compatible staining protocols.

Spectral Considerations:

FITC has excitation/emission maxima at 499/515 nm , which must be considered when selecting additional fluorophores:

Methodological Approach for Multiplexing:

• Panel Design:

  • Begin with FITC-conjugated TKFC antibody as the foundation

  • Add fluorophores with minimal spectral overlap

  • Consider target abundance when assigning fluorophores (use brighter fluorophores for less abundant targets)

• Compensation Controls:

  • Prepare single-stained controls for each fluorophore

  • Include an unstained control and fluorescence minus one (FMO) controls

  • Use compensation beads for consistent and bright signals

• Titration in Multiplex Context:

  • Re-titrate antibodies in the context of the full panel

  • Optimal concentrations may differ in multiplex versus single-stain experiments

• Staining Protocol Harmonization:

  • Ensure all antibodies in the panel are compatible with the same fixation/permeabilization methods

  • If combining surface and intracellular markers, perform surface staining before fixation when possible

• Sequential Staining Strategy:

  • For complex panels, consider sequential staining approaches

  • Example workflow: surface markers → fixation → permeabilization → intracellular markers

• Data Analysis Considerations:

  • Implement proper compensation in analysis software

  • Use visualization tools like biexponential scaling for better display of compensated data

  • Consider advanced analysis methods like tSNE or UMAP for high-dimensional data

This methodological framework enables researchers to effectively incorporate TKFC Antibody, FITC conjugated into multiplex panels while minimizing artifacts and maximizing data quality.

What are common issues encountered with TKFC Antibody, FITC conjugated and their solutions?

When working with TKFC Antibody, FITC conjugated, researchers may encounter several technical challenges. The following table outlines common issues, their potential causes, and recommended solutions:

Additional methodological recommendations include:

• Antibody Validation:

  • Test antibody specificity using positive and negative control samples

  • Confirm staining pattern matches expected subcellular localization of TKFC

  • Consider alternative detection methods (e.g., unlabeled primary with FITC-secondary) if signal is consistently weak

• Protocol Optimization:

  • Document all protocol variations systematically

  • Change only one variable at a time when troubleshooting

  • Prepare fresh buffers regularly to prevent contamination

• Instrument Considerations:

  • Calibrate cytometer or microscope regularly

  • Verify laser alignment and detector sensitivity

  • Consider the impact of different instrument configurations when comparing results

These methodological approaches provide a systematic framework for addressing common technical challenges when working with TKFC Antibody, FITC conjugated.

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

Validating antibody specificity is crucial for ensuring reliable experimental results. For TKFC Antibody, FITC conjugated, researchers should implement a multi-faceted validation strategy:

Control Samples Approach:

• Positive Controls: Test the antibody on samples with confirmed TKFC expression

  • Cell lines with known TKFC expression

  • Tissues with documented TKFC distribution patterns

  • Transfected cells overexpressing TKFC

• Negative Controls: Confirm absence of staining in:

  • Cell lines lacking TKFC expression

  • TKFC knockout or knockdown samples

  • Tissues known to be negative for TKFC expression

Blocking Experiments:

• Pre-incubate the antibody with recombinant TKFC protein before staining

• A specific antibody will show significantly reduced staining after blocking

• Use titrated amounts of blocking protein to demonstrate dose-dependent inhibition

Orthogonal Validation Approaches:

• Multiple Antibody Verification:

  • Compare staining patterns with different anti-TKFC antibodies targeting distinct epitopes

  • Concordant results from different antibodies increase confidence in specificity

• Correlation with Gene Expression:

  • Compare protein detection with mRNA expression data

  • Use RT-PCR, RNA-seq, or in situ hybridization to confirm TKFC expression in samples

• Molecular Weight Verification:

  • Perform Western blot analysis with the unconjugated version of the same antibody

  • Confirm band corresponds to predicted molecular weight of TKFC (~60 kDa)

Methodological Controls for Fluorescence Techniques:

• Isotype Control: Use FITC-conjugated rabbit IgG at the same concentration

• Autofluorescence Control: Examine unstained samples to assess natural fluorescence

• Fluorescence Spillover: Use single-stained controls in multicolor experiments

Documentation and Reporting:

Researchers should maintain detailed records of validation experiments, including:

• Specific lots or batches of antibody used

• Complete experimental conditions

• Images or data from control experiments

• Any limitations identified during validation

This comprehensive validation approach ensures that experimental results with TKFC Antibody, FITC conjugated can be interpreted with confidence and enhances reproducibility across different research settings.

How can TKFC Antibody, FITC conjugated be utilized in studying TKFC's role in cellular antiviral responses?

TKFC has been identified as a repressor of IFIH1-mediated cellular antiviral responses . FITC-conjugated TKFC antibody provides a valuable tool for investigating this important regulatory function. The following methodological approaches can be employed:

Viral Infection Models:

• Temporal Analysis:

  • Track TKFC localization and expression levels before, during, and after viral challenge

  • Use time-course experiments with fixed timepoints to capture dynamic changes

  • Correlate TKFC distribution patterns with viral replication markers

• Co-localization Studies:

  • Combine TKFC Antibody, FITC conjugated with antibodies against viral components

  • Utilize confocal microscopy to assess spatial relationships between TKFC and viral structures

  • Quantify co-localization using statistical measures (e.g., Pearson's correlation coefficient)

IFIH1 Pathway Interaction Analysis:

• Multiplex Immunofluorescence:

  • Simultaneously detect TKFC (FITC), IFIH1, and downstream signaling components

  • Assess correlation between TKFC levels and IFIH1 pathway activation state

  • Create activation/inhibition profiles across different cell types

• Stimulus-Response Experiments:

  • Treat cells with various pathway activators (e.g., poly(I:C))

  • Monitor TKFC redistribution using live-cell imaging with anti-TKFC-FITC

  • Quantify nuclear/cytoplasmic ratios of TKFC during response

Functional Knockdown/Overexpression Studies:

• Expression Level Correlation:

  • Modulate TKFC expression using siRNA, CRISPR, or overexpression constructs

  • Quantify TKFC levels by flow cytometry using TKFC Antibody, FITC conjugated

  • Correlate expression levels with antiviral response metrics

• Rescue Experiments:

  • Reintroduce wild-type or mutant TKFC into knockdown models

  • Use FITC-conjugated antibody to confirm expression and localization

  • Assess functional recovery of antiviral response regulation

Single-Cell Analysis:

• Flow Cytometry:

  • Develop multiparameter panels including TKFC-FITC and viral response markers

  • Identify cell subpopulations with differential TKFC expression

  • Correlate TKFC levels with activation states at single-cell resolution

• Imaging Flow Cytometry:

  • Combine quantitative flow analysis with cellular imaging

  • Assess morphological changes associated with TKFC redistribution

  • Quantify nuclear translocation during antiviral responses

These methodological approaches leverage the specificity and fluorescence properties of TKFC Antibody, FITC conjugated to dissect the role of TKFC in antiviral immunity, potentially revealing novel insights into immunoregulatory mechanisms.

What considerations should be made when using TKFC Antibody, FITC conjugated in quantitative image analysis?

Quantitative image analysis with TKFC Antibody, FITC conjugated requires careful attention to experimental design, image acquisition, and analytical methodology to ensure accurate and reproducible results.

Pre-Acquisition Considerations:

• Sample Preparation Standardization:

  • Maintain consistent fixation, permeabilization, and staining protocols

  • Process all experimental groups in parallel

  • Use the same antibody lot across experiments when possible

• Controls for Quantification:

  • Include calibration samples with known TKFC concentrations

  • Use fluorescent beads to normalize intensity measurements

  • Prepare standard curves when performing absolute quantification

• Minimizing Variability:

  • Control environmental factors (temperature, humidity)

  • Standardize timing between sample preparation and imaging

  • Account for photobleaching through appropriate controls

Image Acquisition Parameters:

• Microscope Settings:

  • Document all acquisition parameters (exposure time, gain, binning)

  • Maintain identical settings across all samples within an experiment

  • Avoid pixel saturation which prevents accurate intensity measurement

• Sampling Strategy:

  • Implement systematic random sampling to avoid selection bias

  • Acquire sufficient fields of view for statistical power

  • Consider 3D acquisition (Z-stacks) for volumetric analysis

• Resolution Considerations:

  • Match pixel size to the scale of structures being analyzed

  • Consider Nyquist sampling criteria for optimal resolution

  • Balance resolution requirements with photobleaching constraints

Analytical Methodology:

• Segmentation Approach:

  • Develop robust algorithms for cellular/subcellular compartment identification

  • Test segmentation accuracy on diverse image sets

  • Implement automated methods to reduce operator bias

• Intensity Measurements:

  • Convert raw intensity to standardized units using calibration samples

  • Account for background fluorescence through appropriate subtraction methods

  • Consider local background correction for heterogeneous samples

• Statistical Analysis:

  • Calculate appropriate descriptive statistics (mean, median, distribution)

  • Implement statistical tests appropriate for your experimental design

  • Account for multiple comparisons when analyzing many parameters

Specialized Analytical Techniques:

Reporting Standards:

For reproducible quantitative imaging with TKFC Antibody, FITC conjugated, document:

• Complete antibody information (source, lot, concentration)

• Detailed sample preparation methodology

• All image acquisition parameters

• Image processing workflows with software versions

• Raw data availability plan

Adherence to these methodological guidelines ensures that quantitative image analysis with TKFC Antibody, FITC conjugated generates reliable and interpretable data.