TRIM5 Antibody, FITC conjugated

What is TRIM5 antibody and what cellular functions does it detect?

TRIM5 antibody detects Tripartite motif-containing protein 5, also known as RING finger protein 88 or RING-type E3 ubiquitin transferase TRIM5. This protein functions as a single protein RING finger E3 ubiquitin ligase with demonstrated antiretroviral activities . The antibody specifically recognizes TRIM5, which belongs to the TRIM/RBCC protein family characterized by a RING domain, B-box zinc finger motifs, and a coiled-coil domain . TRIM5 uniquely contains a B30.2 (also known as PRY-SPRY) C-terminal domain and exists as homotrimers, which are critical for its restriction activity against retroviruses . The FITC-conjugated version of the antibody allows for direct fluorescent detection of TRIM5 in various experimental applications such as flow cytometry and immunofluorescence microscopy.

How should TRIM5 antibody, FITC conjugated be stored to maintain optimal activity?

TRIM5 antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . It's crucial to avoid repeated freeze-thaw cycles as this can significantly reduce antibody performance . The antibody is typically preserved in a storage buffer containing 0.03% Proclin 300 and 50% Glycerol in 0.01M PBS at pH 7.4, which helps maintain stability during storage . For short-term storage of reconstituted antibody (up to one month), 2-8°C under sterile conditions is recommended, while for longer periods (up to six months), storage at -20°C to -70°C under sterile conditions is advised to preserve activity .

What is the specificity of TRIM5 antibody toward different species?

Commercial TRIM5 antibodies vary in their species reactivity. The FITC-conjugated TRIM5 antibody from Cusabio described in the search results has specific reactivity toward human TRIM5, as it was developed using recombinant human Tripartite motif-containing protein 5 (amino acids 1-493) as the immunogen . Different homologs of TRIM5 share varying degrees of sequence homology across species. For example, mouse TRIM5 shares approximately 49% amino acid sequence homology with human TRIM5 and 81% with rat TRIM5 . When designing experiments involving different species, researchers should carefully select antibodies with appropriate species reactivity or validate cross-reactivity experimentally to ensure accurate results.

What are the common applications for TRIM5 antibody, FITC conjugated in HIV research?

TRIM5 antibody, FITC conjugated is particularly valuable in HIV research due to TRIM5's role in retroviral restriction. Common applications include:

• Immunofluorescence microscopy to visualize TRIM5 localization and potential colocalization with viral components like gp120 and autophagy markers like LC3B .

• Flow cytometry to quantify TRIM5 expression levels in different cell populations, such as CD4+ T cells, CD8+ T cells, B cells, and monocytes from HIV-infected patients .

• Investigation of autophagy-mediated viral restriction, as research has shown that TRIM5α directly recognizes HIV-1 and targets it for autophagic destruction .

• Comparative studies between normal progressors (NP), long-term nonprogressors (LTNP), and healthy donors (HD) to understand the relationship between TRIM5 expression and HIV-1 control .

These applications have revealed that higher levels of TRIM5α are associated with better control of HIV-1 infection in long-term nonprogressors, suggesting its potential role in natural resistance to disease progression .

How can researchers optimize triple immunofluorescence protocols using TRIM5 antibody, FITC conjugated with other markers?

Optimizing triple immunofluorescence protocols with TRIM5 antibody, FITC conjugated requires careful consideration of antibody combinations and microscopy settings. Based on published methodologies:

• Antibody Selection: When studying HIV-1 restriction, combine TRIM5 antibody (FITC conjugated) with antibodies against viral proteins (e.g., gp120 or Nef) and autophagy markers (e.g., LC3B). Ensure secondary antibodies are compatible and have minimal spectral overlap .

• Protocol Optimization:

  • Incubate cells with primary antibodies (e.g., rabbit polyclonal anti-TRIM5α at 1:50 dilution) for optimal time and temperature

  • After thorough PBS rinsing, incubate with appropriate fluorophore-conjugated secondary antibodies (e.g., Alexa 594 conjugated goat anti-rabbit IgG at 1:400 dilution)

  • Include proper controls by omitting primary antibodies to assess background staining

• Imaging and Analysis:

  • Use confocal microscopy with appropriate filter sets to minimize bleed-through

  • Quantify colocalization by calculating the percentage of TRIM5α-positive dots that colocalize with viral protein dots and autophagy marker dots

  • Analyze a minimum of 30 cells per sample with multiple independent researchers performing counts to ensure reproducibility

This approach has successfully demonstrated significant colocalization between TRIM5α, HIV-1 proteins, and autophagy markers in LTNP compared to NP, suggesting TRIM5α's involvement in autophagic control of HIV-1 .

What factors affect TRIM5α protein expression levels that might not be reflected in mRNA levels?

Research has revealed interesting discrepancies between TRIM5α protein and mRNA expression levels, particularly in HIV-1 infected patients. Several factors can influence this relationship:

• Post-transcriptional regulation: Despite similar mRNA levels, TRIM5α protein expression varies significantly between patient groups (LTNP, NP, and HD), suggesting important post-transcriptional regulatory mechanisms .

• Protein stabilization mechanisms: In LTNP patients, TRIM5α protein appears to be more stable than in NP patients, potentially due to reduced proteasomal degradation . This aligns with observed higher TRIM5α protein levels despite similar mRNA expression.

• Cell-type specific regulation: Different PBMC subsets show varied patterns of TRIM5α expression. B cells and monocytes from LTNP maintain TRIM5α expression levels similar to healthy donors, while these cell types in NP show significantly decreased expression . This differential regulation occurs independently of mRNA levels.

• Association with autophagy machinery: TRIM5α has been found in complexes with autophagy proteins like ATG14 and AMBRA1, which are components of the BECN1 complex involved in autophagy initiation . These interactions may stabilize TRIM5α protein in specific cellular contexts.

• Translational efficiency: Differences in translation rates or ribosomal loading of TRIM5α mRNA could account for the observed disparities between mRNA and protein levels .

When designing experiments to study TRIM5α function, researchers should consider analyzing both mRNA and protein levels to capture the full regulatory landscape affecting this important restriction factor.

How does human TRIM5α differ from rhesus monkey TRIM5α in experimental HIV restriction assays?

Human and rhesus monkey TRIM5α exhibit significant differences in their anti-HIV-1 restriction capabilities:

• Restriction efficiency: Rhesus monkey TRIM5α demonstrates substantially higher efficiency in blocking HIV-1 infection compared to human TRIM5α . This species-specific difference has important implications for HIV research models and potential therapeutic approaches.

• Mechanism of action: Human TRIM5α appears to be much less efficient in blocking HIV-1 infection than rhesus monkey TRIM5α, specifically in precision autophagy targeting of the HIV-1 capsid . This functional difference may be attributed to variations in the B30.2/SPRY domain that interacts with viral capsids.

• Autophagy dependence: Human TRIM5α restriction efficacy may be more dependent on autophagy processes than rhesus TRIM5α. This has been demonstrated in Langerhans cells, which represent a first line of defense against HIV-1 . The autophagy machinery appears to be required for human TRIM5α to effectively transduce antiviral signaling and establish a protective state against HIV-1 .

• Experimental considerations: When designing HIV restriction assays, these species-specific differences must be considered. Using human TRIM5α in restriction experiments may require additional autophagy modulation or higher expression levels to observe significant effects compared to experiments with rhesus TRIM5α .

These differences highlight the evolutionary adaptations of TRIM5α across primate species and explain why humans remain susceptible to HIV-1 infection despite possessing TRIM5α protein.

What is the relationship between TRIM5α expression, autophagy, and HIV-1 control in different patient populations?

Research examining TRIM5α expression in different HIV-1 patient populations has revealed complex relationships:

• Long-term nonprogressors (LTNP): These patients demonstrate significantly higher levels of TRIM5α-positive cells (>80%) compared to normal progressors (NP, <50%) . This correlates with enhanced autophagic control of HIV-1, as evidenced by increased colocalization of TRIM5α with HIV-1 proteins (gp120, Nef) and autophagy markers (LC3B) in autophagic vacuoles .

• Cell type-specific patterns: Interestingly, the enhanced TRIM5α expression in LTNP is not uniform across all cell types:

  • B cells from LTNP show significantly higher TRIM5α expression compared to NP

  • Monocytes show a positive trend toward higher expression in LTNP

  • CD4+ and CD8+ T cells display similar TRIM5α levels in both LTNP and NP

• Lack of direct correlation with viral load: Analysis revealed no linear correlation between TRIM5α-positive cells and patient viremia, suggesting complex regulatory mechanisms beyond simple viral suppression .

• Autophagy pathway connection: TRIM5α appears to work in concert with other autophagy factors (BECN1, AMBRA1, ATG5) previously shown to be involved in HIV-1 restriction in nonprogressor patients . This suggests a multifaceted autophagy-based restriction mechanism.

This relationship suggests potential therapeutic approaches that might enhance TRIM5α-mediated restriction through autophagy modulation, particularly in specific cell populations like B cells and monocytes that appear critical for natural HIV-1 control in LTNP patients.

What controls should be included when using TRIM5 antibody, FITC conjugated in flow cytometry experiments?

When designing flow cytometry experiments with TRIM5 antibody, FITC conjugated, several controls are essential:

• Isotype control: Include a FITC-conjugated rabbit IgG isotype control at the same concentration as the TRIM5 antibody to identify non-specific binding . This is particularly important as the TRIM5 antibody is polyclonal, which may exhibit higher background compared to monoclonal antibodies.

• Unstained control: Include cells without any antibody to establish autofluorescence baseline and set appropriate gating strategies.

• Fluorescence minus one (FMO) controls: When performing multicolor flow cytometry (e.g., simultaneously analyzing TRIM5 with CD4, CD8, CD14, or CD19 markers), include FMO controls lacking the TRIM5-FITC antibody to properly set boundaries for positive populations.

• Positive and negative biological controls:

  • Use cell types known to express different levels of TRIM5 (e.g., B cells and monocytes from healthy donors as positive controls)

  • Include samples from different subject groups (e.g., healthy donors, long-term nonprogressors, and normal progressors) to establish expression patterns across populations

• Titration experiments: Perform antibody titration to determine optimal concentration for specific signal detection while minimizing background.

These controls ensure accurate interpretation of TRIM5 expression patterns across different cell populations and patient groups, as demonstrated in studies comparing CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD14+ monocytes .

What are the recommended fixation and permeabilization protocols for intracellular staining with TRIM5 antibody, FITC conjugated?

For optimal intracellular staining with TRIM5 antibody, FITC conjugated, the following fixation and permeabilization protocol is recommended:

• Cell preparation:

  • Isolate peripheral blood mononuclear cells (PBMC) using standard density gradient separation

  • Wash cells in PBS and adjust concentration to 1-5 × 10^6 cells/mL

• Fixation:

  • Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

  • Alternative fixatives like 2% formaldehyde can be used but may require protocol optimization

• Washing:

  • Wash cells thoroughly with PBS to remove excess fixative

  • Centrifuge at 300-400×g for 5 minutes and discard supernatant

• Permeabilization:

  • Permeabilize cells using 0.1-0.5% Triton X-100 or a commercial permeabilization buffer

  • Incubate for 10 minutes at room temperature

  • For studying TRIM5α in context of autophagy, saponin-based permeabilization may better preserve autophagic structures

• Blocking:

  • Block with 2-5% normal serum (from the same species as the secondary antibody if using indirect staining) for 30 minutes

• Antibody staining:

  • Incubate with TRIM5 antibody, FITC conjugated at appropriate dilution (typically 1:50-1:100)

  • For co-staining with other intracellular markers (e.g., LC3B, HIV-1 proteins), add respective primary antibodies simultaneously

  • Incubate for 1 hour at room temperature or overnight at 4°C

• Final washes:

  • Wash 3 times with PBS containing 0.1% Tween-20

  • Resuspend in appropriate buffer for analysis

This protocol has been successfully employed to analyze TRIM5α expression in different cell populations and its colocalization with autophagy markers and HIV-1 proteins in patient samples .

How can researchers quantify colocalization between TRIM5α and HIV-1 proteins in immunofluorescence experiments?

Quantifying colocalization between TRIM5α and HIV-1 proteins requires rigorous methodology to ensure accurate and reproducible results:

• Sample preparation:

  • Prepare cells on appropriate slides or coverslips

  • Perform triple immunofluorescence using antibodies against TRIM5α, HIV-1 proteins (e.g., gp120 or Nef), and autophagy markers (e.g., LC3B)

  • Include proper controls by omitting primary antibodies to assess non-specific binding

• Image acquisition:

  • Use confocal microscopy with appropriate filter settings to minimize bleed-through

  • The Leica TCS SP2 confocal microscope has been successfully used for this purpose

  • Acquire z-stack images to ensure complete cellular visualization

  • Maintain consistent exposure and gain settings across all samples

• Quantification methods:

  • Method 1: Calculate the percentage of colocalization between gp120-positive dots that also colocalize with LC3-positive dots and TRIM5α-positive dots relative to total gp120-positive dots

  • Method 2: Use colocalization software plugins (e.g., JACoP for ImageJ) to calculate Pearson's correlation coefficient or Manders' overlap coefficient

  • Method 3: Perform intensity correlation analysis to identify positively correlated pixel intensities

• Statistical analysis:

  • Analyze a minimum of 30 cells per patient to ensure statistical power

  • Have three independent researchers perform cell counting to minimize observer bias

  • Present data as mean ± standard deviation and apply appropriate statistical tests to compare groups

This approach has revealed significantly higher percentages of colocalization between TRIM5α, gp120, and LC3-positive dots in long-term nonprogressors compared to normal progressors (p < 0.0001), providing evidence for TRIM5α's role in autophagic control of HIV-1 .

How might TRIM5 antibody be used to study potential therapeutic approaches targeting TRIM5-mediated restriction?

TRIM5 antibody, FITC conjugated provides valuable tools for investigating therapeutic approaches targeting TRIM5-mediated viral restriction:

• Autophagy modulation strategies: Research suggests that autophagy inducers might enhance TRIM5α's antiviral activity and could potentially be employed in combination with antiretroviral drugs . TRIM5 antibody can be used to monitor changes in TRIM5α levels and localization following treatment with autophagy inducers like rapamycin or physiological inducers.

• Cell-type specific targeting: Studies have shown differential expression of TRIM5α across immune cell subsets, with particularly important roles in B cells and monocytes from long-term nonprogressors . TRIM5 antibody can help identify which cell populations would benefit most from TRIM5-enhancing therapies.

• Protein stabilization approaches: The discrepancy between TRIM5α mRNA and protein levels suggests potential benefits from interventions that increase TRIM5α protein stability . Researchers can use TRIM5 antibody to assess the efficacy of proteasome inhibitors or other compounds that might reduce TRIM5α degradation.

• Mechanistic studies of restriction: TRIM5 antibody enables visualization of interactions between TRIM5α, viral components, and cellular autophagy machinery . This can guide the development of peptides or small molecules that enhance these interactions to promote viral clearance.

• Screening assays: High-throughput screening for compounds that enhance TRIM5α expression or function can utilize FITC-conjugated TRIM5 antibody in flow cytometry or automated microscopy workflows to rapidly identify promising candidates.

These approaches leverage the natural restriction mechanisms observed in HIV-1 controllers and could lead to novel therapeutic strategies that complement traditional antiretroviral approaches.

What are the key considerations when comparing TRIM5α expression between different patient cohorts?

• Cell subset composition: Different PBMC subsets show varied TRIM5α expression patterns . Researchers should either analyze specific cell populations separately (using markers like CD4, CD8, CD14, CD19) or ensure similar cell type distributions across patient samples.

• Protein vs. mRNA analysis: Studies have shown discrepancies between TRIM5α protein and mRNA levels . Both should be analyzed using complementary techniques (western blot/flow cytometry for protein, qPCR for mRNA) to capture the full regulatory landscape.

• Patient characteristics standardization:

  • Duration of infection

  • Antiretroviral treatment history

  • Viral load measurements

  • CD4+ T cell counts

  • Age and sex distribution

• Technical considerations:

  • Consistent sample processing and storage conditions

  • Standardized staining protocols and antibody lots

  • Inclusion of appropriate controls for each experimental batch

  • Blinded analysis to prevent observer bias

• Functional correlation: Beyond quantifying expression levels, assess functional outcomes like colocalization with viral proteins and autophagy markers . This provides insight into TRIM5α's active engagement in viral restriction rather than merely its presence.

• Statistical approach: Ensure sufficient sample sizes for adequate statistical power. When analyzing multiple cell types or parameters, apply appropriate corrections for multiple comparisons to avoid false positives.

These considerations have allowed researchers to identify significant differences in TRIM5α expression between LTNP and NP patients, particularly in B cells and monocytes, despite the absence of a linear correlation with viremia .

What are the emerging roles of TRIM5α in immune responses beyond direct viral restriction?

Beyond its well-established role in direct viral restriction, TRIM5α is emerging as a multifunctional protein in immune responses:

• Antiviral signaling: TRIM5α appears to initiate antiviral signaling pathways, with autophagy machinery serving as a functional platform for this process . This suggests TRIM5α acts not only through direct viral capsid recognition but also by triggering broader immune responses.

• Pattern recognition receptor functions: TRIM5α can function similarly to pattern recognition receptors, sensing viral components and subsequently activating NF-κB and AP-1 signaling pathways . Mouse homologs like TRIM12c have been shown to stimulate Type I IFN and NF-κB pathways along with TNFR-Associated Factor 6 .

• Intersection with autophagy pathways: TRIM5α interacts with autophagy factors including ATG14, AMBRA1, and components of the BECN1 complex . These interactions suggest potential roles in regulating general autophagy processes beyond xenophagy (pathogen-targeted autophagy).

• Immune cell homeostasis: The differential expression of TRIM5α across immune cell subsets, particularly its maintenance in B cells and monocytes from LTNP patients , suggests potential roles in immune cell function and survival during chronic viral infection.

• Inflammation regulation: Recent findings have implicated B cells in chronic constitutive inflammation during HIV infection . TRIM5α's higher expression in B cells from controllers suggests it may play a role in modulating inflammatory responses during chronic infection.

These emerging functions position TRIM5α as a bridge between intrinsic immunity (direct viral restriction) and broader immune responses, making it an attractive target for immunomodulatory approaches beyond direct antiretroviral strategies.

How might technological advances improve detection and functional analysis of TRIM5α using antibody-based approaches?

Emerging technologies promise to enhance TRIM5α detection and functional analysis using antibody-based approaches:

• Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy can provide nanoscale resolution of TRIM5α interactions with viral capsids and autophagy machinery, revealing structural details beyond the diffraction limit of conventional microscopy. This could clarify how TRIM5α recognizes and targets HIV-1 for autophagic degradation .

• Live-cell imaging: Development of minimally disruptive fluorescent tags for TRIM5α, combined with live-cell compatible antibody fragments (nanobodies), could enable real-time visualization of TRIM5α dynamics during viral infection and restriction.

• Mass cytometry (CyTOF): This technology allows simultaneous analysis of >40 parameters at the single-cell level, enabling comprehensive phenotyping of TRIM5α expression across immune cell subsets in conjunction with activation markers, cytokines, and signaling molecules.

• Proximity labeling approaches: Techniques like BioID or APEX2 fused to TRIM5α could identify proximity interactions in living cells, revealing the complete interactome of TRIM5α during viral restriction.

• Single-cell analysis: Combining single-cell RNA sequencing with antibody-based protein detection (CITE-seq) could reveal correlations between TRIM5α protein levels and transcriptional programs at single-cell resolution across diverse immune populations.

• Spatial transcriptomics: Integrating TRIM5 antibody-based detection with spatial transcriptomics could map TRIM5α protein localization in tissues relative to gene expression patterns and viral reservoirs.

These technological advances will provide deeper insights into TRIM5α's role in viral restriction and broader immune functions, potentially identifying new therapeutic targets and approaches for enhancing natural immunity against retroviruses.