TOB1 Antibody, FITC conjugated

What is TOB1 protein and what cellular functions does it perform?

TOB1 (Transducer of ERBB2 1), also known as TOB or TROB1, is a protein with UniprotID P50616 that functions as a transcriptional regulator. It belongs to the BTG/TOB family of antiproliferative proteins that play important roles in cell cycle regulation and tumor suppression. TOB1 interacts with various cellular pathways including those involved in cell proliferation, differentiation, and apoptosis. The protein contains conserved domains that mediate protein-protein interactions, particularly with proteins involved in mRNA deadenylation and degradation processes .

What is the principle behind FITC conjugation in antibodies?

FITC (Fluorescein isothiocyanate) conjugation involves the covalent attachment of the fluorescent FITC molecule to proteins such as antibodies. The isothiocyanate group (-N=C=S) in FITC reacts with primary amine groups on proteins, forming stable thiourea bonds. This reaction typically occurs at alkaline pH conditions (pH 8.0-9.5) where lysine residues are deprotonated. The resulting FITC-antibody conjugate emits bright green fluorescence when excited by blue light (approximately 495 nm), making it detectable in various fluorescence-based applications .

What are the recommended storage conditions for TOB1 Antibody, FITC conjugated?

For optimal stability and performance, TOB1 Antibody, FITC conjugated should be stored at -20°C or -80°C immediately upon receipt. The antibody is supplied in a protective buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation, antibody fragmentation, and loss of FITC fluorescence intensity. For short-term storage (less than one week), the antibody can be kept at 4°C in the dark to prevent photobleaching of the FITC molecule .

What quality control parameters are used to validate TOB1 Antibody, FITC conjugated?

TOB1 Antibody, FITC conjugated undergoes several quality control procedures before commercial release. The antibody is Protein G purified to >95% purity as determined by SDS-PAGE analysis. Functional validation typically includes ELISA testing against the recombinant human TOB1 protein immunogen (amino acids 42-175). Additional quality control may include spectrophotometric analysis to determine the FITC-to-protein ratio, which optimally ranges between 3:1 and 6:1 for most antibody applications. This ensures sufficient fluorescence intensity while maintaining antibody binding capacity .

What is the difference between using TOB1 Antibody, FITC conjugated versus a two-step detection method?

Using TOB1 Antibody, FITC conjugated offers several advantages over two-step detection methods:

• Direct detection eliminates the need for secondary antibodies, reducing protocol time and complexity

• Removes potential sources of non-specific binding from secondary antibodies

• Allows for more precise quantification as the fluorophore-to-antibody ratio is consistent

• Enables multicolor staining protocols with reduced risk of cross-reactivity

How does pH affect the performance of FITC-conjugated antibodies in experimental settings?

The fluorescence properties of FITC are significantly influenced by pH conditions, which researchers must consider when designing experiments. FITC exhibits optimal fluorescence at slightly alkaline pH (7.5-8.5), with emission intensity decreasing substantially below pH 7.0. This pH-dependent behavior is particularly relevant when studying acidic cellular compartments such as endosomes, lysosomes, or tumor microenvironments where lower pH values may result in diminished signal intensity.

Research demonstrates that in acidic tumor microenvironments (pH ≈6.0), FITC-conjugated molecules can show up to a 50% reduction in fluorescence intensity compared to neutral pH conditions. This phenomenon has been exploited in certain pH-sensitive applications, as seen in studies using pH-dependent grafting of cancer cells with antigenic epitopes where cellular fluorescence varies with environmental acidity . When designing experiments with TOB1 Antibody, FITC conjugated, researchers should implement appropriate pH controls and consider alternative fluorophores for strongly acidic environments.

What experimental approaches can be used to study TOB1 protein interactions using FITC-conjugated antibodies?

Several sophisticated methodologies can leverage TOB1 Antibody, FITC conjugated to investigate protein-protein interactions:

• Fluorescence Resonance Energy Transfer (FRET): By pairing TOB1 Antibody, FITC conjugated with antibodies against potential interacting partners labeled with appropriate acceptor fluorophores (e.g., rhodamine), researchers can detect molecular proximities below 10 nm, indicating direct protein interactions.

• Co-localization Studies: High-resolution confocal microscopy using TOB1 Antibody, FITC conjugated alongside antibodies against suspected interaction partners (labeled with spectrally distinct fluorophores) can reveal spatial relationships within cellular compartments.

• Immunoprecipitation Combined with Fluorescence Detection: TOB1 Antibody, FITC conjugated can be used to pull down TOB1 protein complexes, with fluorescence measurements providing quantitative data on complex formation under various experimental conditions.

• Flow Cytometry-Based Protein Interaction Assays: Multi-parameter flow cytometry can simultaneously detect TOB1 (using the FITC-conjugated antibody) and potential interacting proteins, allowing correlation analysis at the single-cell level .

What are the considerations for using TOB1 Antibody, FITC conjugated in multiplex immunofluorescence studies?

Multiplex immunofluorescence studies present several technical challenges when incorporating TOB1 Antibody, FITC conjugated:

• Spectral Overlap Management: FITC has relatively broad emission spectra (peak ~520 nm) that may overlap with other green-yellow fluorophores. Careful fluorophore selection is essential, with fluorophores such as Pacific Blue, Cy5, or APC being ideal partners due to minimal spectral overlap.

• Panel Design Strategy: Position FITC in the panel according to the expected expression level of TOB1. For highly expressed targets, less bright fluorophores like FITC are appropriate, while rare targets benefit from brighter fluorophores.

• Antibody Cross-Reactivity Assessment: Validate that the rabbit-derived TOB1 Antibody, FITC conjugated does not cross-react with other antibodies in the multiplex panel, particularly when using multiple rabbit-derived antibodies.

• Sequential Staining Approaches: For complex multiplex panels, consider sequential staining with intermittent blocking steps to minimize cross-reactivity, particularly when using antibodies from the same host species.

• Compensation Controls: Single-color controls using TOB1 Antibody, FITC conjugated alone are essential for proper spectral unmixing and compensation in flow cytometry or multispectral imaging systems .

How does the polyclonal nature of TOB1 Antibody, FITC conjugated impact experimental design and data interpretation?

The polyclonal nature of TOB1 Antibody, FITC conjugated has significant implications for research applications:

Advantages:

• Recognition of multiple epitopes increases signal strength, especially for low-abundance targets

• Greater tolerance to minor protein modifications or conformational changes

• Often more robust against fixation-induced epitope masking

Experimental Considerations:

• Batch-to-batch variation requires validation when switching lots

• May bind to conserved epitopes present in related proteins, necessitating thorough specificity controls

• Signal intensity can vary across experiments due to heterogeneous binding characteristics

When interpreting data generated with polyclonal TOB1 Antibody, FITC conjugated, researchers should include appropriate controls to account for these characteristics. This includes pre-absorption controls with recombinant TOB1 protein, comparison with monoclonal antibodies when available, and validation across multiple experimental systems to ensure consistency of findings .

What is the optimal protocol for using TOB1 Antibody, FITC conjugated in ELISA applications?

Direct ELISA Protocol for TOB1 Antibody, FITC conjugated:

• Plate Coating:

  • Coat high-binding 96-well plates with capture antigen (recombinant TOB1 or cell lysates) at 1-10 μg/ml in carbonate-bicarbonate buffer (pH 9.6)

  • Incubate overnight at 4°C in a humidified chamber

• Blocking:

  • Wash wells 3 times with PBS + 0.05% Tween-20 (PBST)

  • Block with 2-5% BSA in PBS for 1-2 hours at room temperature

• Antibody Incubation:

  • Dilute TOB1 Antibody, FITC conjugated to 1-10 μg/ml in blocking buffer

  • Add 100 μl to each well and incubate for 2 hours at room temperature protected from light

• Detection:

  • Wash wells 4-5 times with PBST

  • Measure fluorescence using a microplate reader with excitation at 495 nm and emission at 520 nm

• Controls to Include:

  • Blank wells (no antigen, with antibody)

  • Negative control wells (non-relevant protein coating)

  • Concentration curve of recombinant TOB1 protein for quantitation

The sensitivity of this direct fluorescent ELISA typically reaches the low ng/ml range, with a linear dynamic range spanning approximately 2 logs of concentration .

What approaches can researchers use to mitigate photobleaching of FITC when using TOB1 Antibody, FITC conjugated in microscopy?

Photobleaching represents a significant challenge when working with FITC-conjugated antibodies in fluorescence microscopy. The following strategies can effectively minimize this issue:

• Anti-Fade Mounting Media:

  • Use specialized mounting media containing anti-fade agents such as n-propyl gallate, p-phenylenediamine, or commercial formulations

  • Consider mounting media with antioxidants that scavenge free radicals generated during excitation

• Imaging Parameters Optimization:

  • Reduce excitation light intensity to the minimum needed for adequate signal detection

  • Decrease exposure time and increase camera gain when possible

  • Use neutral density filters to attenuate excitation light

• Confocal Microscopy Settings:

  • Narrow the confocal pinhole to reduce out-of-focus light exposure

  • Increase scan speed and apply line or frame averaging to maintain image quality

  • Use resonant scanners for faster imaging with reduced light exposure

• Advanced Microscopy Techniques:

  • Consider using deconvolution algorithms to extract more information from lower-intensity images

  • Implement adaptive illumination strategies that reduce light exposure to previously scanned regions

• Sample Preparation Considerations:

  • Use higher antibody concentrations to achieve stronger initial signals

  • Image samples promptly after staining and store slides at 4°C in the dark when immediate imaging is not possible

What controls should be included when using TOB1 Antibody, FITC conjugated in flow cytometry experiments?

A comprehensive flow cytometry experiment using TOB1 Antibody, FITC conjugated should include the following controls:

• Unstained Cells:

  • Essential for determining autofluorescence levels and setting appropriate voltage parameters

• Isotype Control:

  • Rabbit IgG, FITC conjugated at the same concentration as the TOB1 antibody

  • Controls for non-specific binding due to Fc receptors or hydrophobic interactions

• Single-Color Controls:

  • When performing multicolor experiments, single-color controls are necessary for compensation

  • Especially important when FITC emission overlaps with other fluorophores like PE

• Fluorescence Minus One (FMO) Controls:

  • Include all fluorophores except FITC to determine the boundary between positive and negative populations

  • Critical for accurate gating when analyzing samples with low or variable TOB1 expression

• Positive and Negative Cell Controls:

  • Cell lines with known high or absent TOB1 expression

  • Enables validation of staining protocol and antibody performance

• Blocking Controls:

  • Pre-incubation with unconjugated anti-TOB1 antibody prior to staining with TOB1 Antibody, FITC conjugated

  • Confirms binding specificity to the TOB1 epitope

• Fixation Controls:

  • If fixing cells, compare fixed vs. unfixed samples to assess any fixation-induced fluorescence changes

What are the recommended approaches for validating TOB1 Antibody, FITC conjugated specificity in experimental systems?

Validating antibody specificity is crucial for generating reliable scientific data. For TOB1 Antibody, FITC conjugated, implement these validation approaches:

• Genetic Validation:

  • Compare staining between wild-type cells and TOB1 knockout/knockdown models

  • Observe expected reduction or elimination of signal in cells with reduced TOB1 expression

• Epitope Blocking:

  • Pre-incubate the antibody with recombinant TOB1 protein (particularly the immunogen fragment, amino acids 42-175)

  • Specific binding should be significantly reduced or eliminated

• Orthogonal Detection Methods:

  • Compare results with alternative antibody clones targeting different TOB1 epitopes

  • Correlate findings with mRNA expression data from qPCR or RNA-seq

  • Confirm protein size via Western blot using the same antibody before FITC conjugation

• Signal Localization Assessment:

  • Verify that subcellular localization matches known TOB1 distribution patterns

  • Compare with published literature on TOB1 localization studies

• Cross-Reactivity Evaluation:

  • Test the antibody on cells from different species, particularly if investigating non-human models

  • Evaluate potential cross-reactivity with related family members (e.g., TOB2)

What methods can be used to optimize and troubleshoot immunofluorescence protocols using TOB1 Antibody, FITC conjugated?

When optimizing immunofluorescence protocols with TOB1 Antibody, FITC conjugated, consider the following methodological approaches:

Fixation Optimization:

Permeabilization Strategies:

Troubleshooting Common Issues:

• Weak or Absent Signal:

  • Increase antibody concentration (try 2-5 μg/ml range)

  • Extend incubation time (overnight at 4°C)

  • Try alternative fixation methods that may better preserve the epitope

  • Consider antigen retrieval techniques if using fixed tissues

• High Background:

  • Increase blocking stringency (5-10% normal serum from the same species as secondary antibody)

  • Add 0.1-0.3% Triton X-100 to blocking solution

  • Extend washing steps (5-6 washes of 5-10 minutes each)

  • Reduce antibody concentration or test more diluted preparations

• Photobleaching:

  • Use anti-fade mounting media

  • Minimize exposure during imaging

  • Consider sequential imaging from longest to shortest wavelength fluorophores

• Non-specific Nuclear Staining:

  • Add 10-100 μg/ml RNase to antibody diluent

  • Include 1-5% BSA in blocking buffer to reduce non-specific interactions

How can TOB1 Antibody, FITC conjugated be utilized in cancer research studies?

TOB1 has been implicated in various cancers as a potential tumor suppressor, making TOB1 Antibody, FITC conjugated a valuable tool in cancer research. Researchers can utilize this antibody in several sophisticated applications:

• Cancer Cell Phenotyping:

  • Flow cytometric analysis of TOB1 expression levels across different cancer subtypes

  • Correlation of TOB1 expression with disease progression or treatment response

  • Identification of cancer stem cell populations based on TOB1 expression patterns

• Tumor Microenvironment Studies:

  • Investigation of TOB1 expression in the context of tumor acidosis

  • Leveraging the pH sensitivity of FITC to simultaneously assess microenvironment pH and TOB1 expression

  • Analysis of TOB1 in tumor-associated immune cells and stromal components

• High-Content Screening Applications:

  • Development of image-based assays to screen compounds that modulate TOB1 expression or localization

  • Automated quantification of nuclear versus cytoplasmic TOB1 in response to therapeutic agents

• Circulating Tumor Cell Analysis:

  • Integration of TOB1 staining in liquid biopsy protocols for cancer detection and monitoring

  • Correlation of TOB1 expression patterns with metastatic potential

In particularly innovative approaches, researchers can combine TOB1 Antibody, FITC conjugated with pH-sensitive probes to simultaneously monitor tumor microenvironment conditions and TOB1 expression, as suggested by studies on pH-dependent grafting of cancer cells with antigenic epitopes .

What are the considerations for using TOB1 Antibody, FITC conjugated in live cell imaging experiments?

Live cell imaging with TOB1 Antibody, FITC conjugated presents unique challenges and requires specific considerations:

• Antibody Internalization Dynamics:

  • FITC-conjugated antibodies are not cell-permeable and will only detect surface-exposed epitopes unless delivery systems are employed

  • For intracellular TOB1 detection, consider membrane permeabilization techniques compatible with cell viability

• Physiological Imaging Conditions:

  • Maintain cells in phenol red-free media to reduce background fluorescence

  • Supplement media with antioxidants to reduce phototoxicity

  • Control environmental conditions (pH, temperature, CO₂) to maintain cell health and FITC fluorescence

• Signal-to-Noise Optimization:

  • Implement background subtraction algorithms for quantitative analysis

  • Consider ratiometric imaging approaches to normalize for variations in probe concentration

  • Use appropriate neutral density filters to minimize photobleaching while maintaining adequate signal

• Temporal Resolution Considerations:

  • Balance acquisition frequency with photobleaching concerns

  • Implement intelligent acquisition strategies (e.g., triggered acquisition based on cellular events)

  • Consider the binding kinetics of the antibody when interpreting dynamic events

• Alternative Delivery Strategies:

  • Explore antibody fragments (Fab, scFv) conjugated to FITC for improved tissue penetration

  • Consider cell-penetrating peptide conjugation for intracellular targeting

  • Investigate microinjection techniques for direct delivery of FITC-conjugated antibodies

How can researchers effectively combine TOB1 Antibody, FITC conjugated with other molecular biology techniques?

Integrating TOB1 Antibody, FITC conjugated into multimodal experimental approaches can provide more comprehensive insights:

• Combined Flow Cytometry and Cell Sorting:

  • Use TOB1 Antibody, FITC conjugated to isolate specific cell populations for downstream genomic or proteomic analysis

  • Sort cells based on TOB1 expression levels for functional assays or transcriptome analysis

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Perform pull-down of TOB1 protein complexes using the antibody

  • Utilize fluorescence detection to confirm successful immunoprecipitation before MS analysis

  • Identify novel TOB1 interacting partners under various experimental conditions

• Chromatin Immunoprecipitation (ChIP) Applications:

  • For transcription factor studies, adapt the TOB1 Antibody, FITC conjugated for ChIP protocols

  • Monitor immunoprecipitation efficiency using fluorescence measurements

  • Combine with sequencing (ChIP-seq) to identify TOB1 binding sites genome-wide

• Super-Resolution Microscopy Integration:

  • Exploit the photophysical properties of FITC for techniques like STORM or PALM

  • Achieve nanoscale resolution of TOB1 localization patterns

  • Combine with proximity labeling methods for spatial proteomics applications

• Single-Cell Analysis Pipelines:

  • Use TOB1 Antibody, FITC conjugated in index sorting applications

  • Correlate TOB1 protein levels with single-cell RNA-seq or ATAC-seq data

  • Develop integrated multi-omics approaches to understand TOB1 biology at single-cell resolution

What are the considerations for using TOB1 Antibody, FITC conjugated in primary cell and tissue sample analysis?

Analyzing primary cells and tissue samples with TOB1 Antibody, FITC conjugated requires specific methodological adaptations:

• Tissue-Specific Fixation Optimization:

  • Different tissues may require varied fixation protocols to preserve TOB1 epitopes

  • Consider tissue-specific antigen retrieval methods (heat-induced vs. enzymatic)

  • Validate fixation protocols that maintain both tissue architecture and epitope accessibility

• Autofluorescence Management:

  • Primary tissues often exhibit significant autofluorescence in the FITC channel

  • Implement autofluorescence quenching steps (e.g., Sudan Black B treatment)

  • Consider spectral unmixing approaches to separate FITC signal from autofluorescence

• Antibody Penetration Strategies:

  • For tissue sections over 10 μm, extend antibody incubation times (24-48 hours at 4°C)

  • Consider using antibody penetration enhancers such as Triton X-100 or saponin

  • For whole-mount preparations, implement clearing techniques compatible with immunofluorescence

• Multi-Marker Co-localization:

  • Design marker panels that include cell type-specific identifiers alongside TOB1

  • Implement standardized quantification methods for co-expression analysis

  • Consider sequential staining approaches for complex marker panels

• Validation Across Tissue Types:

  • Confirm antibody performance across different tissue types due to matrix effects

  • Include positive control tissues with known TOB1 expression

  • Develop tissue-specific protocols that account for unique properties (e.g., high lipid content, calcification)

What emerging technologies might enhance research applications of TOB1 Antibody, FITC conjugated?

Several cutting-edge technologies hold promise for expanding the utility of TOB1 Antibody, FITC conjugated in research:

• Spatially Resolved Transcriptomics Integration:

  • Combining TOB1 protein detection with spatial transcriptomics for correlative protein-RNA analysis

  • Development of protocols that preserve both protein epitopes and RNA integrity

  • Creation of multimodal datasets linking TOB1 protein levels to local transcriptional landscapes

• Advanced Microfluidic Applications:

  • Integration of TOB1 staining in organ-on-chip models for real-time monitoring

  • Development of microfluidic antibody delivery systems for improved tissue penetration

  • Creation of high-throughput, low-volume immunoassays for rare sample analysis

• Computational Analysis Enhancements:

  • Implementation of machine learning algorithms for automated TOB1 expression quantification

  • Development of digital pathology tools specific for nuclear protein expression patterns

  • Integration of multiparametric data through advanced computational pipelines

• CRISPR-Based Validation Approaches:

  • Generation of epitope-tagged endogenous TOB1 for antibody validation

  • Development of CRISPR activation/inhibition systems for dynamic TOB1 expression control

  • Creation of reporter systems for real-time monitoring of TOB1 expression

• Novel Conjugation Chemistries:

  • Development of site-specific conjugation methods for improved antibody performance

  • Exploration of photostable FITC derivatives with enhanced brightness and stability

  • Investigation of environmentally-sensitive fluorophores for functional TOB1 studies

The integration of these emerging technologies with traditional antibody-based detection methods will continue to expand our understanding of TOB1 biology and its roles in normal physiology and disease.

How does the field of TOB1 research benefit from fluorescently labeled antibodies?

Fluorescently labeled antibodies, particularly TOB1 Antibody, FITC conjugated, have significantly advanced TOB1 research in several ways:

• Spatial Resolution of TOB1 Biology:

  • Visualization of subcellular distribution patterns has revealed nuclear-cytoplasmic shuttling behaviors

  • Identification of specific nuclear compartments where TOB1 concentrates during different cell cycle phases

  • Detection of TOB1 in previously unappreciated cellular structures

• Quantitative Expression Analysis:

  • Flow cytometric quantification of TOB1 across cell types and disease states

  • Single-cell analysis revealing heterogeneity in TOB1 expression within populations

  • Correlation of expression levels with functional outcomes in various experimental models

• Dynamic Process Investigation:

  • Monitoring of TOB1 translocation in response to signaling events

  • Analysis of protein stability and turnover kinetics through pulse-chase approaches

  • Examination of protein-protein interactions in living systems

• Diagnostic and Therapeutic Applications:

  • Development of TOB1-based biomarkers for disease classification

  • Evaluation of TOB1 as a potential therapeutic target

  • Creation of companion diagnostics for targeted therapies