ICE2 Antibody, FITC conjugated

What is ICE2 Antibody, FITC conjugated, and what are its fundamental properties?

ICE2 Antibody, FITC conjugated is a rabbit polyclonal antibody specifically developed for the detection of human Little elongation complex subunit 2 (ICE2). This antibody has been conjugated to fluorescein isothiocyanate (FITC), a fluorescent dye that allows visualization in fluorescence microscopy and flow cytometry applications. The antibody features excitation/emission wavelengths of 499/515 nm, is compatible with the 488 nm laser line, and is produced using recombinant Human Little elongation complex subunit 2 protein (amino acids 876-982) as the immunogen. The antibody is supplied in liquid form with >95% purity following Protein G chromatography purification .

What are the spectral characteristics and detection parameters for ICE2 Antibody, FITC conjugated?

The spectral properties of ICE2 Antibody, FITC conjugated are as follows:

These spectral characteristics make the antibody compatible with standard fluorescence microscopes, confocal systems, and flow cytometers equipped with 488 nm excitation sources. For optimal detection, ensure instrument settings are calibrated for FITC fluorescence detection .

What are the recommended protocols for using ICE2 Antibody, FITC conjugated in immunofluorescence staining?

For optimal immunofluorescence staining with ICE2 Antibody, FITC conjugated:

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

• Permeabilize with 0.1% Triton X-100 for 10 minutes (for intracellular targets)

• Block with 5% normal serum (from the same species as the secondary antibody) for 1 hour

• Apply diluted ICE2 Antibody, FITC conjugated (optimal dilutions should be determined by the end user) and incubate for 60 minutes at room temperature or overnight at 4°C

• Wash 3× with PBS containing 0.05% Tween-20

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium

Based on studies with other FITC-conjugated probes, tissue staining may achieve maximal fluorescent intensity within 30-60 minutes, which is faster than conventional antibody staining protocols that often require longer incubation times .

How can I optimize detection sensitivity when using ICE2 Antibody, FITC conjugated?

To optimize detection sensitivity with ICE2 Antibody, FITC conjugated:

• Antibody titration: Determine the optimal concentration through a titration series (typically 1:50 to 1:1000) to identify the dilution that provides maximum signal-to-noise ratio

• Extended incubation: While FITC-conjugated probes demonstrate faster staining kinetics than unconjugated antibodies, tissues with lower expression levels may benefit from extended incubation times (60+ minutes), as demonstrated in studies with other FITC-conjugated probes

• Signal amplification: For samples with low target expression, consider using tyramide signal amplification (TSA) systems compatible with FITC

• Photobleaching prevention: Minimize exposure to light during all stages of the experiment; use antifade mounting media containing propyl gallate or n-propyl gallate

• pH optimization: Maintain slightly alkaline conditions (pH 8.0-8.5) during staining procedures to maximize FITC fluorescence output

• Background reduction: Include 0.1-0.3% Triton X-100 in wash buffers to reduce non-specific binding

Researchers should conduct validation experiments to confirm that these optimization strategies maintain antibody specificity while enhancing detection sensitivity .

How can I validate the specificity of ICE2 Antibody, FITC conjugated in my experimental system?

To validate the specificity of ICE2 Antibody, FITC conjugated:

• Positive and negative controls: Use cell lines or tissues with known high and low/no expression of ICE2

• Competitive binding assay: Pre-incubate the antibody with recombinant ICE2 protein (particularly the immunogen sequence 876-982 AA) before staining to demonstrate signal reduction

• siRNA knockdown validation: Compare staining between normal cells and those with ICE2 knockdown

• Co-localization studies: Perform dual staining with another validated ICE2 antibody (using a different fluorophore) to confirm signal overlap

• Western blot correlation: Confirm that relative staining intensity correlates with protein levels detected by Western blot across multiple samples

• Isotype control: Include a FITC-conjugated rabbit IgG isotype control to assess non-specific binding

Applying these validation approaches is essential for establishing confidence in experimental results, particularly when characterizing novel antibodies or when working with complex tissue samples .

What are common artifacts and troubleshooting strategies when using FITC-conjugated antibodies?

Common artifacts and their solutions when working with FITC-conjugated antibodies include:

Additionally, FITC's susceptibility to photobleaching can be mitigated by using image acquisition settings that minimize exposure time while maintaining adequate signal-to-noise ratio .

How can I quantitatively analyze the expression levels of ICE2 using FITC-conjugated antibodies?

For quantitative analysis of ICE2 expression using FITC-conjugated antibodies:

• Standardized image acquisition: Use identical microscope settings (exposure time, gain, offset) for all samples and controls

• Fluorescence calibration: Include calibration beads with known fluorescence intensity to normalize between experiments

• Region of interest (ROI) analysis: Measure integrated fluorescence density within defined cellular compartments using image analysis software

• Background subtraction: Always subtract background signal measured from adjacent negative regions

• Normalization controls: Include housekeeping protein controls or nuclear counterstains for cell number normalization

• Linear dynamic range confirmation: Create a standard curve using samples with known expression levels to verify the linear relationship between signal intensity and protein concentration

Studies with other FITC-conjugated probes have demonstrated excellent linear relationships between fluorescence density measurements and relative protein expression levels determined by other methods. This approach enables semi-quantitative assessment of protein expression across different samples and experimental conditions, particularly valuable for comparative studies .

What are the optimal storage conditions and shelf life for ICE2 Antibody, FITC conjugated?

For optimal preservation of ICE2 Antibody, FITC conjugated:

• Storage temperature: Store at -20°C in small aliquots to minimize freeze-thaw cycles

• Buffer composition: The antibody is supplied in 0.01 M PBS, pH 7.4, containing 0.03% Proclin-300 and 50% glycerol

• Light protection: Store in amber tubes or wrapped in aluminum foil to protect from light exposure

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles; each cycle can reduce activity by 5-10%

• Working solution: Once diluted for use, store at 4°C and use within 24 hours

• Shelf life: Typically 12 months from date of receipt when stored properly at -20°C

The presence of 50% glycerol in the storage buffer helps maintain antibody stability during freeze-thaw cycles by preventing ice crystal formation that could denature the antibody structure. Proclin-300 serves as a preservative to prevent microbial contamination .

How does FITC conjugation impact experimental design compared to other fluorophores?

FITC conjugation influences experimental design in several key ways:

• Spectral considerations: FITC's excitation/emission profile (499/515 nm) may limit multiplex capabilities with other green fluorophores like GFP or BODIPY-FL

• Photobleaching susceptibility: FITC bleaches more rapidly than newer fluorophores like Alexa Fluor 488, requiring careful exposure management

• pH sensitivity: FITC fluorescence decreases significantly below pH 7.0, necessitating pH control during experiments

• Quenching potential: High FITC:protein ratios can cause self-quenching, requiring careful conjugation optimization

• Autofluorescence overlap: FITC emission overlaps with cellular autofluorescence, potentially reducing signal-to-noise ratio in certain tissues

• Alternative considerations: For prolonged imaging or samples with high autofluorescence, consider alternative conjugates like Alexa Fluor 488 or DyLight 488

Understanding these limitations allows researchers to determine whether FITC conjugation is optimal for their specific experimental conditions or whether alternative fluorophores might provide advantages .

What advanced co-localization techniques can be used with ICE2 Antibody, FITC conjugated?

Advanced co-localization approaches for ICE2 Antibody, FITC conjugated include:

• Multi-channel confocal microscopy: Combine with antibodies conjugated to spectrally distinct fluorophores (e.g., Cy3, Cy5) targeting interaction partners

• Super-resolution microscopy: Techniques like STED or STORM can resolve co-localization beyond the diffraction limit

• Förster Resonance Energy Transfer (FRET): When combined with acceptor fluorophore-labeled antibodies against protein interaction partners

• Proximity Ligation Assay (PLA): Combine with another primary antibody to detect proteins in close proximity (<40 nm)

• Fluorescence Lifetime Imaging (FLIM): Measures changes in FITC fluorescence lifetime upon interaction with other molecules

• Quantitative co-localization analysis: Apply Pearson's correlation coefficient, Manders' overlap coefficient, or intensity correlation analysis

Research with other FITC-conjugated probes has demonstrated successful co-localization with antibodies against related proteins, providing insights into protein interactions and cellular distribution patterns. These advanced techniques enable researchers to move beyond simple co-expression analysis to examine functional relationships between ICE2 and other cellular components .

How does ICE2 Antibody, FITC conjugated compare with other methods for detecting ICE2 expression?

ICE2 Antibody, FITC conjugated offers distinct advantages and limitations compared to other detection methods:

Studies with other FITC-conjugated probes have demonstrated that they can achieve comparable sensitivity to conventional two-step antibody staining methods while offering significantly faster processing times. This makes FITC-conjugated antibodies particularly valuable for applications requiring rapid results or high-throughput screening .

What are the advanced research applications of ICE2 Antibody, FITC conjugated in studying protein-protein interactions?

Advanced applications for studying protein-protein interactions include:

• Co-immunoprecipitation validation: Use fluorescence microscopy with ICE2 Antibody, FITC conjugated to confirm co-localization of potential interaction partners identified in co-IP studies

• Live-cell interaction dynamics: Apply ICE2 Antibody, FITC conjugated to permeabilized cells to monitor dynamic interactions during cellular processes (though membrane permeabilization limits truly live-cell applications)

• FRET analysis: Combine with acceptor fluorophore-labeled antibodies against putative interaction partners to measure energy transfer as evidence of proximity

• Protein complementation assays: Use in conjunction with split-reporter systems to visualize and confirm protein interactions

• Correlation with functional assays: Combine immunofluorescence with functional readouts to correlate ICE2 localization with cellular activities

• Quantitative interaction mapping: Apply image analysis algorithms to quantify co-localization coefficients across different cellular compartments or conditions

These advanced applications enable researchers to move beyond simple localization studies to examine functional relationships between ICE2 and other proteins, providing insights into biological mechanisms and potential therapeutic targets .

How can epitope mapping techniques be applied to validate and characterize ICE2 Antibody, FITC conjugated?

Epitope mapping approaches for ICE2 Antibody, FITC conjugated validation include:

• Peptide microarray analysis: Screen binding against overlapping peptides spanning the ICE2 sequence to identify specific recognition sites

• Competitive binding assays: Use synthetic peptides corresponding to different regions of ICE2 to compete for antibody binding

• Mutagenesis approaches: Test antibody binding against ICE2 variants with point mutations or domain deletions

• Hydrogen-deuterium exchange mass spectrometry: Identify regions protected from exchange when the antibody is bound

• X-ray crystallography: Determine the three-dimensional structure of the antibody-antigen complex for complete epitope characterization

• Phage display libraries: Identify mimotopes that interact with the antibody to infer structural epitope characteristics

Similar epitope mapping approaches have been successfully applied to other FITC-conjugated antibodies, enabling precise characterization of binding specificities. For instance, FITC-conjugated cyclic peptides have been mapped to specific recognition domains of integrins, providing insights into their binding mechanisms .