TYW5 Antibody, FITC conjugated

What is TYW5 and why is it significant in cellular research?

TYW5 (tRNA wybutosine-synthesizing protein 5) functions as a tRNA hydroxylase in the wybutosine biosynthesis pathway. This protein catalyzes the hydroxylation of 7-(α-amino-α-carboxypropyl)wyosine (yW-72) into undermodified hydroxywybutosine (OHyW*), which is further transformed into hydroxywybutosine (OHyW) by LCMT2/TYW4. Wybutosine is a hypermodified guanosine with a tricyclic base found at the 3'-position adjacent to the anticodon of eukaryotic phenylalanine tRNA . TYW5's involvement in this post-transcriptional modification pathway makes it an important target for studying tRNA modification processes and their implications in cellular function and disease models. Antibodies against TYW5 enable researchers to visualize and quantify the protein's expression and localization, providing insights into its role in cell biology .

What primary applications are suitable for TYW5 Antibody, FITC conjugated?

TYW5 Antibody, FITC conjugated is particularly suitable for immunofluorescence-based applications. Based on available product information, this conjugated antibody has been validated for:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Flow cytometry (recommended dilution ranges vary by manufacturer)

• Immunocytochemistry/Immunofluorescence microscopy

While Western blot applications are possible with some anti-TYW5 antibodies (at dilutions of 1:100-500), researchers should note that the FITC conjugation may affect detection methods for this application . For immunohistochemistry, optimal dilutions typically range from 1:50-250 (based on a 0.5 mg/ml concentration) . In all applications, researchers should perform preliminary titration experiments to determine optimal antibody concentrations for their specific experimental systems.

How does FITC conjugation impact antibody performance?

FITC (fluorescein isothiocyanate) conjugation introduces a fluorescent marker to the antibody without significantly altering its binding affinity when performed correctly. The conjugation process involves the reaction between FITC and primary amine groups (typically lysine residues) on the antibody molecule . Several factors influence conjugation efficiency and subsequent antibody performance:

• pH significantly affects conjugation efficiency, with optimal labeling occurring at pH 9.5

• Reaction time impacts the fluorescein/protein (F/P) ratio, with maximal labeling achieved in 30-60 minutes at room temperature

• Protein concentration affects conjugation rate, with 25 mg/ml being optimal for efficient labeling

Excessive conjugation (over-labeling) can potentially compromise antibody function by modifying lysine residues in or near the antigen-binding site. Properly conjugated antibodies maintain their immunoreactivity while gaining fluorescence detection capability with excitation/emission at 499/515 nm using a 488 nm laser line .

What are the recommended storage conditions for maintaining TYW5 Antibody, FITC conjugated activity?

Proper storage is crucial for maintaining antibody functionality and fluorophore stability. Based on manufacturer recommendations:

When received, it is advisable to immediately aliquot the antibody into appropriate volumes for single-use to minimize the need for repeated thawing. Always protect FITC-conjugated antibodies from light during handling and storage to preserve fluorescent signal intensity.

How can I optimize TYW5 Antibody, FITC conjugated for flow cytometry applications?

When optimizing TYW5 Antibody, FITC conjugated for flow cytometry, several parameters should be considered:

• Titration of antibody concentration: Perform serial dilutions (starting with manufacturer's recommended dilution) to identify the optimal signal-to-noise ratio. This is particularly important as the recommended dilutions for TYW5 antibodies vary by manufacturer and application .

• Sample preparation: For intracellular staining of TYW5, effective cell permeabilization is critical. Consider using 0.1% saponin or 0.1% Triton X-100 in PBS, with fixation in 2-4% paraformaldehyde prior to permeabilization .

• Compensation settings: When using FITC in multicolor panels, proper compensation is essential due to FITC's relatively broad emission spectrum. FITC's excitation/emission profile (499/515 nm) may overlap with other green fluorophores .

• Dead cell discrimination: Include a viability dye compatible with FITC to exclude non-specific binding to dead cells, which is particularly important for intracellular targets like TYW5.

• Controls: Always include:

  • Unstained cells

  • Fluorescence minus one (FMO) control

  • Isotype control (FITC-conjugated rabbit IgG)

  • Positive control (cell line with confirmed TYW5 expression)

Research has demonstrated successful use of FITC-conjugated antibodies in flow cytometry for analyzing expression patterns in various cell types, as seen in studies examining membrane protein expression in cancer cell lines .

What approaches should be used to validate TYW5 Antibody specificity?

Thorough validation of TYW5 Antibody specificity is essential for reliable experimental results. Recommended validation strategies include:

• Western blot analysis: Though the FITC conjugate may not be optimal for this application, an unconjugated version of the same antibody clone can be used to verify specificity by confirming a single band at the expected molecular weight of TYW5 (approximately 32 kDa) .

• Peptide competition assay: Pre-incubating the antibody with excess immunizing peptide should abolish specific staining in immunofluorescence applications.

• Knockout/knockdown controls: Testing the antibody in samples with genetic deletion or siRNA-mediated knockdown of TYW5 should show reduced or absent signal.

• Orthogonal detection methods: Correlating results from the TYW5 Antibody, FITC conjugated with other detection methods (e.g., qPCR for TYW5 mRNA expression) provides additional validation.

• Cross-species reactivity verification: If using the antibody across multiple species, verify specificity in each species. Some TYW5 antibodies show reactivity to human, mouse, and rat proteins, but this should be experimentally confirmed .

For polyclonal antibodies like the TYW5 rabbit polyclonal, it is particularly important to verify lot-to-lot consistency using these validation approaches .

How does TYW5 Antibody, FITC conjugated perform in multiplex imaging with other fluorophores?

When designing multiplex imaging experiments with TYW5 Antibody, FITC conjugated, consider these technical aspects:

• Spectral characteristics: FITC has excitation/emission peaks at 499/515 nm, making it compatible with standard FITC filter sets and 488 nm laser lines . This spectral profile should be considered when selecting additional fluorophores to minimize spillover.

• Recommended fluorophore combinations:

  • FITC (green) pairs well with:

    • DAPI (blue) for nuclear counterstaining

    • Rhodamine/TRITC (red) for dual labeling

    • Far-red fluorophores (e.g., Cy5) for minimal spectral overlap

• Sequential acquisition: When using confocal microscopy, sequential scanning (rather than simultaneous acquisition) may reduce potential bleed-through between channels.

• Cross-talk considerations: In studies combining FITC-labeled antibodies with other fluorescent proteins, researchers have successfully paired FITC-conjugated antibodies with DAPI nuclear staining in immunohistochemistry applications . This approach has been documented in studies examining neurofilament markers in nerve tissue.

• Antibody panel design: When combining multiple antibodies, consider using primary antibodies from different host species to avoid cross-reactivity in secondary detection systems.

The success of multiplex experiments depends on careful titration of each antibody in the panel and appropriate controls to account for any spillover between channels.

What are common causes of weak signal when using TYW5 Antibody, FITC conjugated?

When experiencing weak signal intensity with TYW5 Antibody, FITC conjugated, investigate these potential causes:

• Antibody concentration: The recommended working dilution varies by application, but typically ranges from 1:50-250 for immunohistochemistry and 1:100-500 for Western blot applications . Insufficient antibody concentration may result in suboptimal detection.

• Target protein expression levels: TYW5 expression may vary across cell types or experimental conditions. Verify expected expression in your experimental system.

• Fixation and permeabilization issues: Overfixation can mask epitopes, while insufficient permeabilization may prevent antibody access to intracellular targets. Consider testing multiple fixation protocols:

  • 4% paraformaldehyde (10-15 minutes)

  • Methanol fixation (-20°C, 10 minutes)

  • 2% paraformaldehyde with 0.1% Triton X-100

• Buffer composition: The antibody is typically supplied in 50% glycerol, 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as preservative . Ensure compatibility with your experimental buffers.

• Fluorophore degradation: FITC is susceptible to photobleaching and pH sensitivity. Storage conditions (multiple freeze-thaw cycles, exposure to light) can diminish fluorescence intensity .

• Detection system sensitivity: Older fluorescence microscopes or flow cytometers may have reduced sensitivity in the FITC channel. Consider instrument optimization or alternative detection methods.

If signal remains weak after addressing these factors, consider consulting with the antibody manufacturer for specific recommendations related to the particular TYW5 antibody product.

How can background fluorescence be reduced when using TYW5 Antibody, FITC conjugated?

Excessive background can significantly impact the signal-to-noise ratio when using FITC-conjugated antibodies. Implement these strategies to minimize background:

• Blocking optimization: Extend blocking time (1-2 hours) using 5-10% serum from the same species as the secondary antibody (if using a detection system) or 1-3% BSA in PBS with 0.1-0.3% Triton X-100 for permeabilization.

• Antibody dilution: Excessive antibody concentration can increase non-specific binding. Perform careful titration experiments to determine optimal concentration .

• Washing procedures: Implement more stringent washing steps (e.g., increase number of washes, duration, or detergent concentration). Consider using PBS with 0.05-0.1% Tween-20 for more effective removal of unbound antibody.

• Autofluorescence reduction:

  • For fixed cells/tissues: Treat with 0.1-1% sodium borohydride or 10-100 mM glycine in PBS prior to blocking

  • For tissues with high autofluorescence: Consider Sudan Black B treatment (0.1-0.3% in 70% ethanol)

• Buffer considerations: Avoid buffers with primary amines (e.g., Tris) which can interfere with FITC conjugation chemistry .

• Sample preparation: Ensure complete removal of fixative before antibody incubation, as residual fixative can increase background fluorescence.

Researchers should systematically test these approaches to identify the optimal conditions for their specific experimental system when working with TYW5 Antibody, FITC conjugated.

What is the recommended protocol for cell permeabilization when using TYW5 Antibody, FITC conjugated?

Since TYW5 is an intracellular protein involved in tRNA modification, effective permeabilization is crucial for antibody access. Based on successful immunostaining approaches for intracellular targets:

• For cultured cells (recommended protocol):

  • Fix cells with freshly prepared 4% paraformaldehyde in PBS (pH 7.4) for 15 minutes at room temperature

  • Wash 3× with PBS (5 minutes each)

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS for 10 minutes at room temperature

  • Wash 3× with PBS (5 minutes each)

  • Block with 3% BSA in PBS with 0.1% Triton X-100 for 1 hour at room temperature

  • Incubate with TYW5 Antibody, FITC conjugated at optimal dilution (typically 1:50-250) overnight at 4°C

  • Wash 5× with PBS (5 minutes each)

  • Mount with anti-fade mounting medium containing DAPI for nuclear counterstaining

• Alternative permeabilization agents:

  • 0.5% saponin in PBS (gentler permeabilization, may better preserve morphology)

  • 100% ice-cold methanol (-20°C, 10 minutes; combines fixation and permeabilization)

  • 0.1% SDS in PBS (more stringent permeabilization for difficult epitopes)

• For tissue sections:

  • After deparaffinization and antigen retrieval, permeabilize with 0.2% Triton X-100 in PBS for 15-20 minutes

  • Longer permeabilization time (up to 30 minutes) may be necessary for thicker sections

The optimal permeabilization method should be empirically determined for each cell type and experimental system, as over-permeabilization can disrupt cellular morphology while insufficient permeabilization can limit antibody accessibility.

What controls are essential when designing experiments with TYW5 Antibody, FITC conjugated?

Robust experimental design requires appropriate controls to ensure valid interpretation of results. When working with TYW5 Antibody, FITC conjugated, incorporate these controls:

• Negative controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration as the TYW5 antibody

  • Secondary-only control (if using secondary detection system)

  • Unstained sample to establish autofluorescence baseline

  • Peptide competition/blocking control: Pre-incubation of the antibody with excess immunizing peptide

• Positive controls:

  • Cell lines or tissues with confirmed TYW5 expression

  • Recombinant TYW5 protein (the antibody was raised against recombinant human TYW5 protein, amino acids 1-152)

• Technical controls:

  • Fluorescence minus one (FMO) controls for multicolor flow cytometry

  • Spectral compensation controls when performing multiplex imaging

• Biological controls:

  • TYW5 knockdown or knockout samples (if available)

  • Comparative analysis across tissue or cell types with varying TYW5 expression levels

These controls help distinguish specific from non-specific binding and ensure that observed signals represent genuine TYW5 protein localization and expression.

How can I quantitatively analyze TYW5 expression using FITC-conjugated antibodies?

Quantitative analysis of TYW5 expression using FITC-conjugated antibodies can be approached through several methodologies:

• Flow cytometry quantification:

  • Measure median fluorescence intensity (MFI) of the FITC channel

  • Calculate fold change relative to control samples

  • For absolute quantification, consider using calibration beads with known quantities of FITC molecules

• Quantitative microscopy approaches:

  • Measure integrated density of FITC signal in defined regions of interest

  • Perform colocalization analysis with organelle markers to determine subcellular distribution

  • Use software like ImageJ/FIJI, CellProfiler, or QuPath for automated image analysis

• Statistical considerations:

  • Normalize FITC signal to cell number or nuclear counterstain

  • Analyze sufficient cells (typically >100 cells per condition) for statistical power

  • Apply appropriate statistical tests based on data distribution

• Quantification controls:

  • Include samples with known TYW5 expression levels for calibration

  • Maintain consistent imaging parameters (exposure time, gain settings) across all samples

  • Account for background fluorescence and autofluorescence in quantification

For robust quantification, researchers should also consider the excitation/emission properties of FITC (499/515 nm) and ensure appropriate laser lines (488 nm) and filter sets are used for optimal detection sensitivity .

How does fixation method affect the performance of TYW5 Antibody, FITC conjugated?

The choice of fixation method significantly impacts epitope preservation, antibody accessibility, and retention of fluorescence signal when using TYW5 Antibody, FITC conjugated:

• Paraformaldehyde fixation:

  • 2-4% paraformaldehyde (PFA) in PBS for 10-20 minutes at room temperature

  • Preserves cell morphology and is compatible with FITC fluorescence

  • May require additional permeabilization step for intracellular targets like TYW5

  • Benefits: Maintains protein localization well; compatible with most antibodies

• Methanol fixation:

  • 100% ice-cold methanol at -20°C for 10 minutes

  • Simultaneously fixes and permeabilizes cells

  • May enhance detection of some nuclear proteins

  • Caution: Can reduce FITC fluorescence intensity; may extract membrane lipids

• Glutaraldehyde fixation:

  • 0.1-0.5% glutaraldehyde mixed with PFA

  • Provides stronger fixation for structural proteins

  • Caution: May cause higher autofluorescence; can mask epitopes through extensive cross-linking

• Acetone fixation:

  • Ice-cold acetone for 5-10 minutes

  • Rapidly dehydrates and fixes cells

  • Caution: May disrupt membrane structures; can extract lipids

What are the considerations for using TYW5 Antibody, FITC conjugated in different cell and tissue types?

When applying TYW5 Antibody, FITC conjugated across different biological samples, consider these critical factors:

• Cell type-specific considerations:

  • Expression levels: TYW5 expression varies across cell types; adjust antibody concentration accordingly

  • Autofluorescence: Certain cells (e.g., macrophages, hepatocytes) exhibit higher autofluorescence that may interfere with FITC detection

  • Permeabilization requirements: Different cell types may require adjusted permeabilization protocols based on membrane composition

• Tissue-specific considerations:

  • Antigen retrieval: Formalin-fixed paraffin-embedded tissues require optimized antigen retrieval (typically heat-induced epitope retrieval in citrate buffer, pH 6.0, or EDTA buffer, pH 9.0)

  • Section thickness: Optimize antibody penetration by using appropriate section thickness (typically 5-10 μm)

  • Tissue autofluorescence: Tissues containing collagen, elastin, or lipofuscin require additional steps to quench autofluorescence

• Species cross-reactivity:

  • The TYW5 antibody shows reactivity to human, mouse, and rat proteins

  • Sequence homology should be verified when using in other species

  • Validate antibody performance in each species independently

• Technical adaptations:

  • Fresh vs. fixed samples: Fresh or lightly fixed samples may require different antibody concentrations than heavily fixed tissues

  • Penetration time: Increase antibody incubation time for tissue sections (24-48 hours at 4°C) compared to cell monolayers

  • Washing stringency: Tissue sections typically require more extensive washing to remove unbound antibody

Researchers should conduct preliminary validation studies when applying the TYW5 Antibody, FITC conjugated to new experimental systems or tissue types.

How can I integrate TYW5 research with broader cellular pathway analysis?

TYW5's role in tRNA modification connects it to broader cellular processes that can be investigated through integrated experimental approaches:

• Pathway integration strategies:

  • Co-immunoprecipitation using non-conjugated TYW5 antibodies to identify protein interaction partners

  • Dual immunofluorescence with TYW5 Antibody, FITC conjugated and antibodies against related tRNA modification enzymes (e.g., LCMT2/TYW4)

  • Correlation of TYW5 expression with tRNA modification levels through mass spectrometry analysis

• Functional studies:

  • Knockdown/knockout of TYW5 followed by assessment of wybutosine modification status

  • Analysis of phenotypic changes in protein translation efficiency or accuracy

  • Cell stress response studies to determine how TYW5 function is affected under various cellular stresses

• Disease relevance investigation:

  • Compare TYW5 expression and localization in normal versus disease tissues

  • Correlate TYW5 function with known pathways disrupted in specific diseases

  • Examine post-translational modifications of TYW5 that may regulate its activity

• Multi-omics integration:

  • Correlate TYW5 protein expression (via antibody-based methods) with transcriptomic data

  • Integrate with metabolomics to examine effects on tRNA modification metabolites

  • Combine with ribosome profiling to assess impact on translation

These integrated approaches enable researchers to position TYW5 within its broader biological context and understand how this tRNA modification enzyme contributes to cellular homeostasis and potential disease mechanisms.