TRIM65 Antibody, FITC conjugated

Basic Research Questions

• What is TRIM65 and why would researchers use a TRIM65 antibody?

TRIM65 is an E3 ubiquitin ligase belonging to the TRIM/RBCC protein family. In humans, it's a 517 amino acid protein with a mass of 57.4 kDa primarily localized in the cytoplasm . It contains a characteristic tripartite motif with a RING-finger domain (responsible for E3 ligase activity), a B-box domain, a coiled-coil domain, and a SPRY domain .

Researchers use TRIM65 antibodies to study its diverse biological functions, including:

• Regulation of miRNA pathways through TNRC6 protein ubiquitination

• Modulation of autophagy processes

• Inhibition of apoptosis (particularly in intestinal ischemia/reperfusion injury)

• Roles in innate immunity and inflammation

TRIM65 antibodies enable detection in various applications including Western blotting, ELISA, immunocytochemistry, immunofluorescence, and flow cytometry .

• What is FITC conjugation and why is it useful for TRIM65 antibody applications?

FITC (Fluorescein isothiocyanate) conjugation involves the chemical attachment of FITC molecules to antibodies through reaction with free amino groups, forming stable thiourea bonds . This process creates directly detectable antibodies without requiring secondary detection steps.

FITC provides several advantages for TRIM65 antibody applications:

• High quantum efficiency (brightness)

• Excitation maximum at 495 nm and emission maximum at 525 nm (green fluorescence)

• Compatibility with standard fluorescence microscopy filters and flow cytometers

• Direct detection without secondary antibodies, reducing background and cross-reactivity

• Facilitation of multicolor staining with spectrally distinct fluorophores

• What are the main applications of TRIM65 antibody, FITC conjugated?

FITC-conjugated TRIM65 antibodies serve various research applications:

• How does TRIM65 function as an E3 ubiquitin ligase?

TRIM65 functions as an E3 ubiquitin ligase through its RING-finger domain, which facilitates the transfer of ubiquitin from E2 enzymes to substrate proteins. The functional mechanism involves:

• Recognition and binding of target substrates through its B-box, coiled-coil, and SPRY domains

• Recruitment of ubiquitin-loaded E2 enzymes via its RING domain

• Catalysis of ubiquitin transfer to lysine residues on target proteins

• Promotion of either monoubiquitination or polyubiquitination with specific chain topologies

Research has established that TRIM65 predominantly mediates K48-linked polyubiquitination of targets like TNRC6 proteins and TOX4, marking them for proteasomal degradation . Studies using TRIM65 mutants with disrupted RING domains demonstrate that this catalytic activity is essential for its biological functions .

• What cellular pathways involve TRIM65?

TRIM65 participates in multiple cellular pathways through its E3 ubiquitin ligase activity:

Intermediate Research Questions

• What are the key considerations when designing experiments with FITC-conjugated TRIM65 antibodies?

When designing experiments with FITC-conjugated TRIM65 antibodies, researchers should consider:

• Fixation method compatibility: FITC fluorescence is pH-sensitive, with optimal performance at pH 8-9. Fixation protocols should maintain appropriate pH.

• Signal intensity and photobleaching: FITC is moderately susceptible to photobleaching. Use anti-fade mounting media, minimize light exposure, and optimize acquisition settings.

• Tissue autofluorescence: FITC's emission spectrum overlaps with cellular autofluorescence. Include proper controls and background subtraction.

• Epitope accessibility: Ensure fixation and permeabilization methods don't mask the TRIM65 epitope recognized by the antibody.

• Antibody concentration optimization: Perform titration experiments to determine optimal antibody concentration (typically between 1:10-50 dilution for immunofluorescence) .

• Spectral overlap considerations: When combining with other fluorophores, select those with minimal spectral overlap with FITC for multi-color experiments.

• Validation controls: Include TRIM65 knockout/knockdown samples or blocking peptides to confirm specificity of the observed FITC signal.

• How do I optimize FITC-conjugated TRIM65 antibody concentration for immunofluorescence assays?

Optimizing FITC-conjugated TRIM65 antibody concentration involves a systematic titration approach:

• Begin with the manufacturer's recommended dilution range (typically 1:10-50 for immunofluorescence) .

• Prepare a dilution series spanning this range (e.g., 1:5, 1:10, 1:25, 1:50, 1:100) using identical samples known to express TRIM65.

• Maintain consistent protocols for fixation, permeabilization, blocking, and imaging parameters across all samples.

• Evaluate results based on signal-to-noise ratio rather than absolute signal intensity—optimal concentration provides strong specific signal with minimal background.

• Include negative controls (isotype control antibody at identical concentration, secondary antibody only, and if available, TRIM65-knockout cells).

• Consider differential expression levels—TRIM65 is widely expressed but levels vary across tissues, potentially requiring different antibody concentrations .

• For quantitative studies, ensure the selected concentration falls within the linear range of detection.

• Document the optimized concentration and standardize it across related experiments to ensure reproducibility.

• What controls should be used when working with FITC-conjugated TRIM65 antibodies?

Appropriate controls for FITC-conjugated TRIM65 antibody experiments include:

• How can I validate the specificity of a FITC-conjugated TRIM65 antibody?

Validating FITC-conjugated TRIM65 antibody specificity requires multiple complementary approaches:

• Western blot analysis: Confirm the antibody detects a single band at the expected molecular weight (57.4 kDa for human TRIM65) .

• Genetic knockdown/knockout: Compare staining patterns in wild-type versus TRIM65-depleted samples—specific signal should be substantially reduced .

• Competitive blocking: Pre-incubation with the immunizing peptide should eliminate specific staining.

• Expected localization pattern: TRIM65 should show primarily cytoplasmic distribution with potential nuclear co-localization depending on context. Immunofluorescence confocal microscopy has shown TRIM65 can co-localize with its substrates both in the cytoplasm and nucleus .

• Comparative analysis with multiple antibodies: Use antibodies targeting different TRIM65 epitopes and compare staining patterns .

• Heterologous expression: Compare staining in cells transfected with TRIM65 expression vectors versus empty vector controls.

• Cross-reactivity testing: If working with non-human samples, verify specificity in the species of interest (TRIM65 orthologs exist in mouse, rat, bovine, and other species) .

Advanced Research Questions

• How can I use FITC-conjugated TRIM65 antibodies to study the relationship between TRIM65 and TNRC6 proteins in miRNA regulation?

To investigate TRIM65's relationship with TNRC6 proteins in miRNA regulation:

• Co-immunofluorescence: Combine FITC-conjugated TRIM65 antibodies with differently labeled antibodies against TNRC6 proteins to assess co-localization in P-body-like structures where miRNA silencing occurs .

• Proximity ligation assay (PLA): Use FITC-conjugated TRIM65 antibody with TNRC6 antibodies to visualize direct interactions, producing fluorescent spots where proteins are within 40nm.

• Time-course experiments: Monitor changes in TRIM65 and TNRC6 protein co-localization following perturbations to the miRNA pathway. Research has shown that TRIM65 overexpression results in degradation of endogenous TNRC6A protein when proteasome inhibitors are absent .

• Sucrose gradient fractionation with immunofluorescence: Analyze FITC-labeled TRIM65 distribution across fractions containing different RISC complexes. Studies have shown TRIM65 sediments in low-density sucrose fractions with a subset of TNRC6 and AGO proteins .

• Reporter assays: Correlate the degree of FITC-TRIM65 and TNRC6 co-localization with functional outcomes using miRNA reporter systems like let-7a activity assays. Research has demonstrated that silencing TRIM65 reduces let-7a reporter activity, consistent with its role in regulating miRNA activity .

• How can FITC-conjugated TRIM65 antibodies be used to study TRIM65's role in intestinal ischemia/reperfusion injury models?

FITC-conjugated TRIM65 antibodies can be valuable for studying TRIM65's role in intestinal ischemia/reperfusion (II/R) injury:

• Expression pattern analysis: Track TRIM65 downregulation in hypoxia-reoxygenation (H/R) treated intestinal epithelial cells and II/R-induced intestinal tissue using quantitative immunofluorescence. Research has shown TRIM65 is significantly decreased in these conditions .

• Colocalization with TOX4: Visualize the spatial relationship between FITC-labeled TRIM65 and differently labeled TOX4 (a TRIM65 substrate in II/R injury) in both cytoplasmic and nuclear compartments. Studies have demonstrated that TRIM65 binds directly to the N-terminal of TOX4 through its coiled-coil and SPRY structural domains .

• Timeline studies: Monitor TRIM65 expression, localization, and interaction with TOX4 across different timepoints during ischemia and subsequent reperfusion phases.

• Comparative wild-type versus knockout analysis: Use FITC-TRIM65 antibodies in parallel with apoptosis markers to correlate TRIM65 expression with cellular outcomes. Research has shown TRIM65 knockout mice exhibit markedly aggravated intestinal apoptosis and II/R injury .

• Mechanistic analysis: Correlate TRIM65 distribution with ubiquitination activity. Studies have demonstrated that TRIM65 mediates the K48 ubiquitination and degradation of TOX4 depending on its E3 ubiquitin ligase activity, thereby inhibiting H/R-induced intestinal epithelial apoptosis .

Troubleshooting Questions

• Why might I observe non-specific binding with my FITC-conjugated TRIM65 antibody and how can I reduce it?

Non-specific binding with FITC-conjugated TRIM65 antibodies can result from several factors:

• How can I address weak fluorescence signal issues when using FITC-conjugated TRIM65 antibodies?

To address weak fluorescence signal with FITC-conjugated TRIM65 antibodies:

• Antibody concentration adjustment: Increase antibody concentration while monitoring for non-specific binding. Start with recommended dilutions for immunofluorescence (typically 1:10-50) .

• Antigen retrieval optimization: For formalin-fixed tissues, test different antigen retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0 or Tris-EDTA pH 9.0).

• Fixation method modification: Compare different fixatives (paraformaldehyde, methanol, acetone) as TRIM65 epitope detection may vary with fixation method.

• pH optimization: Ensure buffers are at pH 8-9 where FITC fluorescence is optimal .

• Permeabilization enhancement: Test increased detergent concentrations or alternative permeabilization agents (0.5% Triton X-100, 0.1% saponin) to improve antibody access to intracellular TRIM65.

• Improved microscopy settings: Use appropriate filter sets optimized for FITC (excitation ~495nm, emission ~525nm), increase exposure time or detector gain while monitoring background.

• Storage and handling considerations: FITC-conjugated antibodies are light-sensitive—minimize exposure to light during storage and processing to prevent photobleaching before imaging .

• How should I store FITC-conjugated TRIM65 antibodies to maintain optimal activity?

Proper storage of FITC-conjugated TRIM65 antibodies is critical for maintaining their activity:

• Temperature conditions: Store at -20°C for long-term storage in small aliquots to prevent repeated freeze-thaw cycles, which can cause protein denaturation and fluorophore degradation .

• Light protection: FITC is highly susceptible to photobleaching—store in amber vials or wrap containers in aluminum foil to protect from light exposure .

• Aliquoting strategy: Upon receipt, divide into single-use aliquots to avoid repeated freezing and thawing of the entire stock.

• Buffer considerations: Most commercial FITC-conjugated antibodies contain stabilizers and preservatives like sodium azide—avoid introducing contaminants during handling .

• Working dilution storage: Diluted working solutions can be stored at 4°C for short periods (1-2 weeks) but with reduced signal intensity over time—prepare fresh dilutions for optimal results.

• Quality control: Before each critical experiment, verify antibody performance using positive control samples with known TRIM65 expression to detect any deterioration in antibody performance.

• What are common fixation artifacts when using FITC-conjugated TRIM65 antibodies in immunofluorescence, and how can they be avoided?

Common fixation artifacts with FITC-conjugated TRIM65 antibodies include:

• Epitope masking: Chemical fixatives can alter protein conformation, obscuring the TRIM65 epitope. Compare different fixatives (4% paraformaldehyde, methanol, acetone) to determine optimal epitope preservation.

• Autofluorescence induction: Aldehyde-based fixatives can create autofluorescent products. Use paraformaldehyde instead of glutaraldehyde or quench aldehyde groups with 50mM NH₄Cl post-fixation.

• Over-fixation: Excessive fixation duration or concentration can reduce antibody penetration. Optimize fixation time (typically 10-20 minutes for 4% paraformaldehyde).

• Membrane permeabilization inconsistencies: Insufficient permeabilization prevents antibody access to intracellular TRIM65, while excessive permeabilization may extract cytoplasmic proteins. Optimize detergent concentration and duration.

• pH-induced fluorescence variations: FITC fluorescence is pH-dependent. Maintain consistent buffer pH (ideally 7.4-8.0) during fixation, staining, and mounting .

• Nuclear shrinkage or cytoplasmic extraction: Can distort TRIM65 distribution patterns. Use fixatives that preserve cellular morphology.

To minimize artifacts, develop a standardized protocol with optimized fixative, concentration, duration, temperature, and post-fixation processing specific for TRIM65 detection.