TTC14 Antibody, FITC conjugated

What is TTC14 Antibody, FITC conjugated and what are its primary applications?

TTC14 Antibody, FITC conjugated is a rabbit polyclonal antibody targeting Tetratricopeptide repeat protein 14 (also known as TPR repeat protein 14 or KIAA1980) that has been labeled with fluorescein isothiocyanate (FITC) . This antibody specifically recognizes the human TTC14 protein and is primarily used in research applications including:

• Immunofluorescence microscopy

• Flow cytometry (particularly intracellular applications)

• Tissue cross-reactivity (TCR) studies

• ELISA assays (when specified by manufacturer)

The antibody is typically generated using recombinant human TTC14 protein fragments (often amino acids 569-770) as the immunogen . This reagent is strictly for research use only and not intended for diagnostic or therapeutic procedures .

What are the spectral characteristics of FITC conjugation and which detection systems are most compatible?

FITC is a small organic molecule that exhibits the following spectral properties when conjugated to antibodies:

• Excitation maximum: approximately 495-499 nm

• Emission maximum: approximately 515-525 nm

• Laser line compatibility: 488 nm (typically from argon lasers)

This spectral profile makes FITC-conjugated antibodies compatible with:

• Flow cytometers equipped with 488 nm lasers

• Fluorescence microscopes with FITC filter sets

• Plate readers with appropriate excitation/emission capabilities

FITC has remained one of the most commonly used fluorescent dyes for flow cytometry analysis due to its brightness and widespread instrument compatibility . The quantum yield of FITC remains relatively high after conjugation, contributing to its continued popularity in research applications .

What are the recommended storage and handling conditions for TTC14 Antibody, FITC conjugated?

To maintain optimal activity of TTC14 Antibody, FITC conjugated:

Following these guidelines will help preserve both the antibody's binding capacity and the fluorescence intensity of the FITC conjugate over time.

How does the FITC-labeling index affect binding affinity and specificity of TTC14 antibody?

The FITC-labeling index (number of FITC molecules per antibody) significantly impacts antibody performance. Research has demonstrated an inverse correlation between FITC-labeling index and binding affinity:

• Optimal range: 3-6 FITC molecules per antibody molecule

• Over-conjugation effects:

  • Decreased binding affinity for target antigen

  • Increased non-specific binding

  • Reduced solubility

  • Internal quenching (reduced brightness)

A study specifically examining FITC-labeled antibodies found that "the FITC-labeling index in antibody was negatively correlated with the binding affinity for its target antigen. Immunohistochemically, an antibody with a higher labeling index had a tendency to be more sensitive, but was also more likely to yield non-specific staining" .

For optimal results with TTC14 Antibody, FITC conjugated, researchers should select preparations with appropriate labeling indices to balance detection sensitivity with specific binding.

What methodological considerations should be taken when using TTC14 Antibody, FITC conjugated in flow cytometry?

When designing flow cytometry experiments with TTC14 Antibody, FITC conjugated, consider the following methodological factors:

• Cell Preparation:

  • For intracellular staining, proper fixation and permeabilization are essential as TTC14 is an intracellular target

  • Example protocol: "Intracellular flow cytometric analysis of permeabilized cells labeling TTC14 using ab166905 at 1/100 dilution"

• Blocking and Reducing Background:

  • Implement appropriate blocking: "Add 2 mL of blocking solution (PBS containing 10% fetal bovine serum [FBS]) and incubate for 20 minutes at room temperature to reduce non-specific binding"

• Antibody Dilution Optimization:

  • Optimal dilutions should be determined experimentally for each application

  • Typical starting dilution for flow cytometry: 1:100 to 1:500

• Controls:

  • Include appropriate isotype controls (rabbit IgG-FITC negative control)

  • Unstained controls for setting baseline fluorescence

  • Single-color controls if performing multicolor analysis

• Compensation:

  • FITC has spectral overlap with other fluorophores like PE, requiring proper compensation when used in multicolor panels

What is the recommended protocol for conjugating FITC to TTC14 antibodies in a laboratory setting?

For researchers preparing their own FITC-conjugated TTC14 antibodies, the following protocol outline is recommended:

Materials Required:

• Purified TTC14 antibody (preferably >95% pure)

• High-quality FITC (fluorescein isothiocyanate)

• Carbonate-bicarbonate buffer (0.1M, pH 9.5)

• PBS (pH 7.4)

• Sephadex G-25 or equivalent desalting column

Protocol Steps:

• Antibody Preparation:

  • Ensure antibody concentration is at least 2 mg/ml in carbonate-bicarbonate buffer

  • Remove any amine-containing substances (e.g., Tris) or sodium azide by dialysis against carbonate-bicarbonate buffer

• Conjugation Reaction:

  • Dissolve FITC in anhydrous DMSO at 1 mg/ml

  • Add FITC solution to antibody at various FITC:protein ratios (typically 10-20 μg FITC per mg of antibody)

  • Incubate for 30-60 minutes at room temperature in the dark with gentle mixing

  • For maximal labeling: "Maximal labelling was obtained in 30–60 minutes at room temperature, pH 9.5 and an initial protein concentration of 25 mg/ml"

• Purification:

  • Separate conjugated antibody from free FITC using a desalting column equilibrated with PBS

  • Collect fractions and analyze by spectrophotometry

• Characterization:

  • Calculate the F/P (fluorescein/protein) ratio using the formula:
    $\text{F/P ratio} = \frac{A_{495} \times \text{MW of IgG}}{195,000 \times A_{280} - (0.35 \times A_{495})}$

  • Optimal F/P ratio: 3-6

• Storage:

  • Add glycerol to 50% final concentration and store at -20°C in small aliquots

How can researchers troubleshoot non-specific binding issues with TTC14 Antibody, FITC conjugated?

When encountering non-specific binding with TTC14 Antibody, FITC conjugated, implement the following troubleshooting strategies:

• Evaluate FITC Labeling Index:

  • Higher labeling indices are associated with increased non-specific staining

  • Consider using preparations with lower F/P ratios for improved specificity

• Optimize Blocking Protocol:

  • Increase blocking time (30-60 minutes)

  • Use alternative blocking reagents (5-10% normal serum from the same species as secondary antibody)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Adjust Antibody Concentration:

  • Perform titration experiments to determine optimal concentration

  • Use the minimum effective concentration that yields positive staining

• Modify Buffer Composition:

  • Add 0.5-1% BSA to reduce background

  • Consider adding 0.05-0.1% Tween-20 to washing buffers

• Implement Additional Controls:

  • Pre-adsorption controls with recombinant TTC14 protein

  • Secondary-only controls to assess secondary antibody background

  • Isotype controls at matching concentrations

How does TTC14 Antibody, FITC conjugated compare to other detection methodologies in immunoassays?

When comparing TTC14 Antibody, FITC conjugated to other detection methodologies:

For applications requiring direct visualization of TTC14 in cellular contexts, FITC-conjugated antibodies offer the advantage of direct detection without additional reagents, though potentially at the cost of some binding affinity .

What approaches can be used to validate the specificity of TTC14 Antibody, FITC conjugated?

To validate the specificity of TTC14 Antibody, FITC conjugated:

• Western Blot Validation:

  • Confirm presence of a single band at the expected molecular weight (~88 kDa)

  • Compare staining pattern across multiple relevant cell lines (e.g., Jurkat, Daudi, Ramos)

• Competitive Inhibition Tests:

  • Pre-incubate antibody with recombinant TTC14 protein before staining

  • Observe reduction or elimination of specific signal

• siRNA Knockdown Controls:

  • Compare staining in cells with and without TTC14 knockdown

  • Expect significantly reduced signal in knockdown cells

• Comparison with Alternative Antibody Clones:

  • Compare staining pattern with other validated anti-TTC14 antibodies

  • Concordant results increase confidence in specificity

• Cross-reactivity Assessment:

  • Test antibody on samples from species other than intended target

  • Verify reactivity aligns with sequence homology predictions

• Flow Cytometry Validation:

  • "Intracellular flow cytometric analysis of permeabilized Jurkat cells labeling TTC14 using ab166905 at 1/100 dilution (red) or a rabbit IgG negative (green)"

What are the optimal experimental conditions for using TTC14 Antibody, FITC conjugated in tissue cross-reactivity studies?

For tissue cross-reactivity (TCR) studies with TTC14 Antibody, FITC conjugated:

• Tissue Preparation:

  • Use freshly frozen or appropriately fixed tissues (typically 10% neutral buffered formalin)

  • Optimize fixation time based on tissue type

  • For frozen sections, maintain 4-8 μm thickness for optimal antibody penetration

• Antibody Selection Criteria:

  • "A FITC-labeled antibody used as a primary antibody in a TCR study should be carefully selected from several differently labeled antibodies to minimize the decrease in the binding affinity and achieve the appropriate sensitivity"

  • Balance FITC labeling index with binding affinity

• Antigen Retrieval:

  • May be required for formalin-fixed tissues

  • Test multiple antigen retrieval methods if initial staining is weak

• Blocking and Staining Protocol:

  • Implement robust blocking: 10% normal serum + 1% BSA in PBS for 30-60 minutes

  • Optimal antibody dilution typically in 1:50 to 1:200 range

  • Incubate overnight at 4°C for maximum sensitivity and specificity

• Controls:

  • Include both positive and negative control tissues

  • Use isotype control at matching concentration

  • Include absorption controls where feasible

• Signal Evaluation:

  • Document signal intensity, localization pattern, and background

  • Compare to known TTC14 expression patterns in literature

What are the latest methodological advances in conjugation chemistry applicable to TTC14 antibodies?

Recent advances in antibody conjugation chemistry applicable to TTC14 antibodies include:

• Site-Specific Conjugation Methods:

  • "Most of these approaches are based on the introduction of mutated residues that serve as targets for site-directed conjugation. Examples include THIOMABs, which carry additional exposed cysteine residues for maleimide conjugation"

  • These approaches produce more homogeneous conjugates with preserved binding affinity

• Enzymatic Conjugation Strategies:

  • "Antibodies can be engineered with recognition sequences that enable spontaneous or enzyme-based conjugation by means of inteins, SNAP, sortase, or transglutaminase"

  • Transglutaminase-mediated conjugation: "The enzymatic linkage between glutamine and lysine side chains by transglutaminase is a key approach... to conjugate Fc donor modules with payloads in a site-specific manner"

• Format Chain Exchange Technology (FORCE):

  • "Antibody derivatives with exchange-enabled Fc-heterodimers are combined with payload-conjugated Fc donors, and subsequent chain-exchange transfers payloads to antibody derivatives in different formats"

  • This allows generation of matrices with consistent drug-to-antibody ratios

• pH-Sensitive Dye Conjugation:

  • Incorporation of pH-sensitive dyes (pHAb) to monitor internalization and trafficking

• Multi-Payload Conjugation:

  • Development of strategies allowing attachment of combinations of payloads

  • Enables simultaneous tracking and functional studies

These advanced methodologies could potentially be applied to TTC14 antibodies to create reagents with superior performance characteristics and functional capabilities.

How can researchers optimize the use of TTC14 Antibody, FITC conjugated in multiplexed immunofluorescence applications?

For optimal multiplexed immunofluorescence using TTC14 Antibody, FITC conjugated:

• Panel Design Considerations:

  • Position FITC in appropriate fluorophore hierarchy (typically early in panel due to brightness)

  • Account for FITC spectral overlap with other fluorophores (particularly PE, PE-Cy5)

  • Balance panel with brightest fluorophores on least abundant targets

• Compensation Strategy:

  • Prepare single-color controls for proper compensation matrix calculation

  • Consider computational approaches to spectral unmixing for highly complex panels

• Sequential Staining Approach:

  • For co-localization studies, consider sequential staining protocols:

    1. First antibody application and detection

    2. Blocking of first antibody binding sites

    3. Application of TTC14 Antibody, FITC conjugated

  • This reduces the risk of cross-reactivity between antibodies

• Antibody Validation:

  • Validate each antibody individually before combining in multiplex panels

  • Test for potential interference between antibodies

• Image Acquisition Settings:

  • Optimize exposure times for each channel to prevent bleed-through

  • Use sequential scanning in confocal microscopy to minimize crosstalk

• Data Analysis:

  • Implement appropriate controls for accurate quantification

  • Consider computational approaches for co-localization analysis

What are the experimental considerations when studying TTC14 protein dynamics using FITC-conjugated antibodies?

When investigating TTC14 protein dynamics using FITC-conjugated antibodies:

• Live Cell Imaging Limitations:

  • FITC-conjugated antibodies require cell permeabilization for intracellular targets like TTC14

  • Not suitable for live cell imaging of endogenous TTC14

• Fixation Timing Experiments:

  • Design time-course experiments with different fixation timepoints

  • Fix cells at defined intervals following stimulus/treatment

  • Stain with TTC14 Antibody, FITC conjugated to capture temporal dynamics

• Quantitative Analysis Approaches:

  • Use digital image analysis software to quantify:

    • Signal intensity (protein expression levels)

    • Subcellular localization patterns

    • Co-localization with organelle markers

• Photobleaching Considerations:

  • Account for FITC photobleaching in time-series acquisitions

  • Consider using anti-fade mounting media with DABCO or similar agents

  • Implement acquisition protocols that minimize exposure times

• Alternative Approaches for Live Dynamics:

  • For live studies, consider:

    • TTC14-fluorescent protein fusion constructs

    • Complementary techniques like FRAP or FLIP with tagged TTC14

How does buffer composition affect the performance of TTC14 Antibody, FITC conjugated in different applications?

Buffer composition significantly impacts the performance of TTC14 Antibody, FITC conjugated:

For optimal results:

• For immunofluorescence: PBS pH 7.4 with 1% BSA and 0.05% Tween-20

• For flow cytometry: PBS pH 7.4 with 1-2% FBS or BSA

• For storage: PBS pH 7.4 with 50% glycerol and 0.03% Proclin 300

How can TTC14 Antibody, FITC conjugated be integrated into high-content screening workflows?

Integration of TTC14 Antibody, FITC conjugated into high-content screening (HCS) workflows:

• Automated Protocol Development:

  • Optimize staining protocols for robotic liquid handling systems

  • Standardize cell seeding density, fixation, and antibody concentrations

  • Validate reproducibility across multiple plates and batches

• Multiparametric Readouts:

  • Combine TTC14 detection with:

    • Nuclear markers (DAPI/Hoechst)

    • Cytoskeletal markers (phalloidin)

    • Functional readouts (apoptosis, proliferation markers)

  • Develop algorithms for feature extraction and pattern recognition

• Quality Control Metrics:

  • Implement Z'-factor calculations to assess assay quality

  • Include positive and negative controls on each plate

  • Develop normalization strategies for plate-to-plate variation

• Data Analysis Pipeline:

  • Establish automated image analysis workflows for:

    • TTC14 expression levels

    • Subcellular localization patterns

    • Morphological correlations

  • Implement machine learning approaches for complex phenotype classification

• Validation Strategies:

  • Confirm hits using orthogonal methods

  • Implement dose-response confirmation assays

  • Correlate image-based findings with biochemical assays

What are the current limitations of TTC14 Antibody, FITC conjugated and how might these be addressed in future formulations?

Current limitations and future improvement strategies for TTC14 Antibody, FITC conjugated:

Future directions may include:

• Development of recombinant monoclonal versions with consistent production quality

• Implementation of site-specific conjugation technologies for optimal FITC positioning

• Creation of TTC14 antibody fragments with enhanced tissue penetration capabilities

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

What novel techniques might be employed to study TTC14 protein interactions using FITC-conjugated antibodies?

Innovative approaches for studying TTC14 protein interactions using FITC-conjugated antibodies:

• Proximity Ligation Assays (PLA):

  • Combine TTC14 Antibody, FITC conjugated with antibodies against putative interaction partners

  • Implement secondary proximity probes to generate amplified fluorescent signals at interaction sites

  • Quantify discrete interaction events in situ

• FRET-Based Interaction Studies:

  • Pair TTC14 Antibody, FITC conjugated (donor) with acceptor-labeled antibodies against interaction partners

  • Monitor energy transfer efficiency as measure of molecular proximity

  • Requires careful spectral overlap consideration and controls

• Lattice Light-Sheet Microscopy Applications:

  • Employ advanced 3D imaging to track TTC14 interactions with reduced phototoxicity

  • Capture rapid dynamic interactions in volumetric datasets

  • Analyze spatial-temporal patterns of interaction events

• Correlative Light and Electron Microscopy (CLEM):

  • Localize TTC14 using FITC-conjugated antibodies by fluorescence microscopy

  • Process same samples for electron microscopy to visualize ultrastructural context

  • Correlate protein localization with detailed subcellular structures

• Expansion Microscopy Integration:

  • Physically expand samples after TTC14 antibody labeling

  • Achieve super-resolution imaging using standard microscopy equipment

  • Map protein interactions with nanoscale precision

• Microfluidic Antibody Capture:

  • Create microfluidic devices coated with TTC14 Antibody, FITC conjugated

  • Capture interaction partners from cell lysates

  • Identify interactions using downstream proteomic analysis