FICD Antibody, FITC conjugated

What is FICD protein and why is it significant in research?

FICD (FIC domain-containing protein) is also known as Adenosine monophosphate-protein transferase FICD, AMPylator FICD, De-AMPylase FICD, Huntingtin yeast partner E, Huntingtin-interacting protein 13 (HIP-13), or Huntingtin-interacting protein E. The protein (UniprotID: Q9BVA6) possesses both AMPylation and de-AMPylation enzymatic activities that play crucial roles in protein folding and endoplasmic reticulum homeostasis . Its interaction with Huntingtin protein makes it particularly relevant in neuroscience research, especially in studies related to Huntington's disease pathogenesis and potential therapeutic approaches .

What are the spectral properties of FITC conjugation and how does it benefit research applications?

FITC (fluorescein isothiocyanate) is a fluorochrome dye with excitation and emission peak wavelengths at approximately 495nm and 525nm, respectively. When excited with ultraviolet or blue light, FITC emits a visible yellow-green fluorescence. The spectral characteristics make it compatible with standard fluorescence microscopy filter sets and flow cytometry configurations . The conjugation process is relatively straightforward and generally does not alter the biological activity of the labeled protein, making it an excellent choice for antibody labeling in various immunological detection methods .

What are the recommended applications for FICD Antibody, FITC conjugated?

• Immunofluorescence microscopy

• Flow cytometry

• Immunohistochemistry on frozen sections

• Surface labeling experiments

A typical flow cytometry application protocol for FITC-conjugated antibodies involves:

• Cell fixation and permeabilization

• Blocking with appropriate buffer

• Primary antibody incubation

• Washing steps to remove unbound antibody

• Analysis using 488nm laser excitation and 530/30 bandpass filter

What controls should be included when designing experiments with FICD Antibody, FITC conjugated?

For rigorous experimental design, the following controls are essential:

Proper implementation of these controls enables accurate interpretation of experimental results and helps distinguish between specific signal and background noise .

What are the optimal storage conditions for maintaining FICD Antibody, FITC conjugated activity?

FICD Antibody, FITC conjugated should be stored at -20°C or -80°C to maintain its activity . The antibody is provided in a buffer containing 50% glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative . To preserve fluorescence intensity and antibody binding capacity:

• Aliquot the antibody upon first thawing to minimize freeze-thaw cycles

• Protect from continuous exposure to light, as FITC is susceptible to photobleaching

• Avoid repeated freeze-thaw cycles that can denature the antibody and reduce binding efficiency

• When working with the antibody, keep it on ice and in amber tubes or wrapped in aluminum foil

When following these guidelines, the antibody can maintain its activity for the duration of its shelf life.

What sample preparation methods optimize detection with FICD Antibody, FITC conjugated?

Effective sample preparation is crucial for optimal antibody binding and signal detection:

For cellular samples:

• Fixation: 4% paraformaldehyde (10-15 minutes) preserves structure while maintaining antigen accessibility

• Permeabilization: 0.1-0.5% Triton X-100 (5-10 minutes) for intracellular targets

• Blocking: 5-10% normal serum or 1-3% BSA (30-60 minutes) to reduce non-specific binding

• Antibody dilution: Start with manufacturer's recommended concentration (typically 1:50-1:200 for immunofluorescence)

• Incubation: Overnight at 4°C or 1-2 hours at room temperature in a humidified chamber

• Washing: Multiple PBS washes between steps to remove unbound antibody

For tissue sections:

• Use frozen sections rather than paraffin-embedded tissues when possible, as paraffin processing may mask epitopes

• Include antigen retrieval steps if using fixed tissues

• Extend blocking time to reduce autofluorescence

These optimizations help maintain FICD epitope integrity while minimizing background fluorescence .

How can researchers address weak or absent signal when using FICD Antibody, FITC conjugated?

When encountering weak or absent signals, consider the following systematic troubleshooting approach:

Additionally, the use of signal amplification systems such as tyramide signal amplification (TSA) may enhance detection sensitivity when working with low-abundance targets .

What strategies can minimize photobleaching of FITC-conjugated antibodies during imaging?

FITC is susceptible to photobleaching, which can compromise experiment reproducibility and quantification. Implement these strategies to preserve fluorescence:

• Add anti-fade reagents to mounting media (e.g., p-phenylenediamine or commercial products like ProLong Gold)

• Reduce exposure time and light intensity during imaging

• Use neutral pH mounting media, as FITC fluorescence is optimal at pH 7.4-8.0

• Image FITC channels first in multi-color experiments

• Employ confocal microscopy with appropriate pinhole settings to reduce out-of-focus light exposure

• Consider using computational approaches such as deconvolution to enhance signal from lower exposure images

• Store slides at -20°C in the dark when not imaging to preserve fluorescence for repeated viewing

These approaches collectively minimize photobleaching while maintaining adequate signal-to-noise ratios for high-quality imaging and analysis .

How can FICD Antibody, FITC conjugated be incorporated into multiplex immunofluorescence studies?

FITC-conjugated antibodies can be effectively combined with other fluorophores in multiplex studies by following these guidelines:

• Spectral compatibility planning: Select companion fluorophores with minimal spectral overlap such as:

  • FITC (excitation: 495nm, emission: 525nm)

  • TRITC/Rhodamine (excitation: 557nm, emission: 576nm)

  • Cy5 (excitation: 650nm, emission: 670nm)

• Sequential detection protocol:

  • Apply antibodies in order of decreasing sensitivity

  • Include blocking steps between antibody applications

  • Consider tyramide signal amplification for low-abundance targets

• Cross-reactivity prevention:

  • Use antibodies raised in different host species

  • Employ directly conjugated primary antibodies when possible

  • Implement Fab fragments to block cross-reactivity between secondary antibodies

• Anti-FITC antibody strategy: Utilize anti-FITC antibodies conjugated to alternative fluorophores for signal amplification or when double-labeling if one antibody is only available as a FITC conjugate .

• Instrumentation considerations: Ensure microscope/flow cytometer is equipped with appropriate filter sets to distinguish between fluorophores, and implement spectral unmixing algorithms for overlapping emissions .

What are the current research applications of FICD antibodies in neuroscience?

FICD antibodies have emerged as valuable tools in neuroscience research, particularly in studying:

• Huntington's disease mechanisms: FICD (also known as HIP13/Huntingtin-interacting protein 13) is being investigated for its interaction with Huntingtin protein and potential role in disease pathogenesis .

• ER stress responses: FICD's AMPylation activity regulates BiP/GRP78 chaperone function during ER stress, which is particularly relevant in neurodegenerative diseases characterized by protein misfolding.

• Neuronal protein quality control: Researchers are exploring FICD's role in maintaining proteostasis in neurons, which are particularly vulnerable to protein aggregation.

• Synaptic plasticity: Emerging evidence suggests potential roles for FICD in regulating protein modifications at synapses.

• Neurodevelopmental processes: FICD's enzymatic activities may influence protein folding crucial for neuronal development and circuit formation.

The FITC conjugation enables direct visualization of FICD localization in neuronal compartments, co-localization with interaction partners, and activity-dependent changes in expression or distribution. These applications highlight FICD as a significant research target in understanding neurological disease mechanisms .

What are the key methodological differences between using FICD Antibody, FITC conjugated and unconjugated FICD antibodies?

The FICD Antibody, FITC conjugated has at least 3 moles of fluorescein per mole IgG as determined spectrophotometrically, providing adequate signal for direct detection methods. The conjugate's purity (>95%, Protein G purified) ensures minimal non-specific binding .

How does antibody cross-reactivity influence experimental design with FICD Antibody, FITC conjugated?

Cross-reactivity considerations are essential for experimental design with FICD antibodies:

• Species cross-reactivity: The FICD Antibody is immunized against human FICD protein (specifically amino acids 63-185) and shows reactivity with human samples. For studies using other species, cross-reactivity should be empirically tested .

• Binding specificity verification:

  • Western blot analysis using recombinant FICD and cell lysates

  • Immunoprecipitation followed by mass spectrometry

  • Competitive binding assays with recombinant FICD protein

  • Knockout/knockdown validation in cellular systems

• Epitope mapping considerations: The antibody targets amino acids 63-185 of human FICD, which may influence detection of splice variants or post-translationally modified forms of the protein .

• Buffer compatibility: The antibody formulation (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300) should be considered when designing experiments, as certain buffers may influence binding specificity .

A methodical approach to validating specificity ensures reliable experimental outcomes and properly accounts for potential cross-reactivity issues when investigating FICD biology.