TIMELESS Antibody, Biotin conjugated

What is the TIMELESS protein and why is it a significant research target?

TIMELESS (also known as TIM, TIM1, hTIM) is a 138.7 kDa nuclear protein consisting of 1208 amino acid residues that belongs to the Timeless protein family. It is widely expressed across tissues including brain, heart, lung, liver, skeletal muscle, kidney, placenta, pancreas, spleen, thymus, and testis . The protein plays critical roles in multiple cellular processes including DNA replication control, maintenance of replication fork stability, genome stability throughout normal DNA replication, DNA repair, and regulation of the circadian clock . Its multifunctional nature makes it a significant target for research across fields from chronobiology to cancer research and virology.

What applications are most suitable for biotin-conjugated TIMELESS antibodies?

Biotin-conjugated TIMELESS antibodies are particularly valuable for applications requiring signal amplification or multiple detection systems. The primary applications include:

The biotin-streptavidin system provides flexibility in detection methods while maintaining high specificity for the TIMELESS protein across multiple experimental platforms .

How should researchers validate the specificity of biotin-conjugated TIMELESS antibodies?

Validation of biotin-conjugated TIMELESS antibodies requires a multi-faceted approach:

• Molecular weight confirmation: Verify detection of the expected 140-150 kDa band in Western blot, corresponding to the TIMELESS protein .

• Subcellular localization: Confirm nuclear localization pattern in immunofluorescence assays, consistent with TIMELESS's known function .

• Positive and negative controls: Include tissues/cells with known TIMELESS expression (HEK-293, NIH/3T3, A549, HeLa, Jurkat, RAW 264.7) as positive controls, and consider using TIMELESS-knockdown samples as negative controls .

• Cross-reactivity assessment: Test against multiple species if cross-reactivity is claimed (human and mouse reactivity is commonly reported) .

• Blocking experiments: Perform pre-incubation with the immunizing peptide to confirm specific binding.

These validation steps are essential for ensuring experimental reproducibility and reliable interpretation of results across different research applications .

What are the optimal sample preparation protocols for detecting TIMELESS using biotin-conjugated antibodies?

Sample preparation protocols should be tailored to both the specific application and the cellular compartment being analyzed:

For Western Blot applications:

• Nuclear extraction is critical as TIMELESS is primarily localized in the nucleus

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors to preserve post-translational modifications

• Heat samples at 95°C for 5 minutes in reducing buffer

• Load 20-40 μg of nuclear protein extract per lane

For Immunofluorescence/Immunohistochemistry:

• For FFPE tissues: Recommended antigen retrieval with TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

• For cell preparations: 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization

• Block with 5% normal serum corresponding to the species of the secondary antibody

• Optimal antibody dilution range: 1:50-1:500 for IF; 1:20-1:200 for IHC

These protocols maximize detection sensitivity while minimizing background, particularly important when using biotin-conjugated antibodies that may exhibit higher background due to endogenous biotin in some tissues .

What controls should be included when using biotin-conjugated TIMELESS antibodies in cell cycle studies?

When studying TIMELESS in the context of cell cycle regulation, the following controls are essential:

Experimental Controls:

• Cell cycle synchronization validation: Include flow cytometry analysis of propidium iodide-stained cells to confirm cell cycle phase distributions

• Avidin/streptavidin-only controls: To assess endogenous biotin background

• Isotype controls: Biotin-conjugated antibodies of the same isotype but irrelevant specificity

• Cell cycle markers co-staining: Include established markers such as cyclin B1 (G2/M), PCNA (S phase)

• Knockdown/knockout validation: TIMELESS siRNA or CRISPR-edited cells as negative controls

Biological Reference Points:

• Asynchronous populations for baseline TIMELESS expression

• G1/S transition cells (where TIMELESS activity increases)

• S-phase cells (peak TIMELESS expression)

• Cells treated with replication stress inducers (e.g., hydroxyurea, UV) to observe TIMELESS recruitment to stalled replication forks

This comprehensive control strategy ensures that observations of TIMELESS dynamics throughout the cell cycle are specific and biologically relevant .

How can researchers optimize dual-labeling experiments involving biotin-conjugated TIMELESS antibodies?

Optimizing dual-labeling experiments with biotin-conjugated TIMELESS antibodies requires careful planning to avoid cross-reactivity and signal interference:

Strategic Approaches:

• Sequential detection: Apply primary antibodies sequentially rather than simultaneously

• Blocking endogenous biotin: Pre-block with avidin/biotin blocking kit before antibody application

• Species selection: Choose primary antibodies from different species for co-labeling partners

• Fluorophore selection: Select fluorophores with minimal spectral overlap when using streptavidin-fluorophore conjugates

Technical Optimization for Dual Immunofluorescence:

• If co-labeling with TIPIN (a known TIMELESS partner), use rabbit anti-TIMELESS biotin-conjugated with mouse anti-TIPIN

• For nuclear co-localization studies, consider:

  • Pre-extraction steps to remove soluble nuclear proteins

  • Super-resolution microscopy techniques for better spatial resolution of colocalizing factors

  • 3D deconvolution to improve visualization of nuclear structures

Controls for Dual Labeling:

• Single-labeled samples for each antibody to establish spectral profiles

• Fluorescence minus one (FMO) controls to assess bleed-through

• Competition assays with unlabeled antibodies to confirm specificity

These optimization strategies improve the reliability of co-localization data when studying TIMELESS interactions with replication fork components or circadian clock proteins .

How does biotin-conjugated TIMELESS antibody performance compare in detecting distinct TIMELESS isoforms?

Biotin-conjugated TIMELESS antibodies may exhibit variable detection capabilities for different TIMELESS isoforms, which is an important consideration for comprehensive functional studies:

When designing experiments to distinguish between isoforms:

• Select antibodies raised against epitopes present in all isoforms for comprehensive detection

• For isoform-specific detection, epitope mapping is crucial to ensure specificity

• Consider the use of 2D gel electrophoresis followed by Western blotting for complex samples

• Validation using overexpression systems of specific isoforms provides important controls

Understanding the epitope location relative to known isoform variations is critical when interpreting research findings, particularly in studies of circadian regulation where isoform expression may vary with circadian timing .

What are the optimal approaches for studying TIMELESS-TIPIN interactions using biotin-conjugated antibodies?

The TIMELESS-TIPIN complex plays a critical role in replication fork stability, making it an important target for advanced research. When using biotin-conjugated TIMELESS antibodies to study this interaction:

Co-immunoprecipitation (Co-IP) Optimization:

• Cross-linking strategy: Consider using membrane-permeable crosslinkers (e.g., DSP) at 0.5-2 mM for 30 minutes to stabilize transient interactions

• Buffer selection: Use buffers containing 150-300 mM NaCl with 0.1% NP-40 to maintain complex integrity while minimizing non-specific binding

• Bead selection: Streptavidin-coated magnetic beads offer advantages over agarose for cleaner results with biotin-conjugated antibodies

Proximity Ligation Assay (PLA) Approach:

• Combine biotin-TIMELESS antibody with rabbit/mouse anti-TIPIN antibody

• Use streptavidin-oligonucleotide conjugate and secondary antibody-oligonucleotide conjugate

• This allows visualization of protein-protein interactions within 40 nm distance in intact cells

Functional Analysis:

• Couple interaction studies with replication stress induction (hydroxyurea treatment)

• Monitor checkpoint activation (CHEK1 phosphorylation) in parallel

• Compare findings in normal vs. stress conditions to assess functional significance

These methodologies enable detailed investigation of how the TIMELESS-TIPIN complex functions in both normal DNA replication and under conditions of replication stress, providing insights into genome stability mechanisms .

How can researchers effectively employ biotin-conjugated TIMELESS antibodies in ChIP-seq experiments?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) with biotin-conjugated TIMELESS antibodies requires specific optimization to study TIMELESS binding to chromatin:

Protocol Optimization:

• Crosslinking parameters: Use 1% formaldehyde for 10 minutes, as over-crosslinking may mask the TIMELESS epitope

• Sonication conditions: Optimize to achieve chromatin fragments of 200-500 bp

• Pre-clearing strategy: Include a streptavidin pre-clearing step to reduce background from endogenous biotinylated proteins

• Elution method: Consider using competitive biotin elution rather than harsh elution buffers to preserve protein-DNA interactions

Specialized ChIP-seq Approaches:

• Sequential ChIP (biotin-TIMELESS followed by TIPIN or replication factors) to identify co-occupancy sites

• DRIP-ChIP (DNA:RNA hybrid immunoprecipitation followed by TIMELESS ChIP) to investigate TIMELESS association with R-loops

• ChIP-seq after synchronization at different cell cycle phases to map temporal chromatin association patterns

Data Analysis Considerations:

• Compare TIMELESS binding sites with known origins of replication

• Analyze co-localization with G-quadruplex structures, which TIMELESS helps replicate past

• Integrate with replication timing data to identify early vs. late replication domains

These approaches enable mapping of TIMELESS chromatin association in relation to its roles in DNA replication and genome stability, providing insights into its function beyond protein-protein interactions .

How can biotin-conjugated TIMELESS antibodies be utilized to investigate its role in thrombogenesis in COVID-19?

Recent research has identified TIMELESS as a key gene mediating thrombogenesis in COVID-19, offering a novel application for biotin-conjugated TIMELESS antibodies:

Experimental Approaches:

• Immunohistochemistry of patient tissues:

  • Compare TIMELESS expression in lung and vascular tissues from COVID-19 patients with and without thrombotic complications

  • Co-stain with markers of endothelial damage and coagulation cascade activation

• Flow cytometric analysis of blood samples:

  • Quantify TIMELESS expression in peripheral blood mononuclear cells from COVID-19 patients

  • Correlate with D-dimer levels and other coagulation parameters

• Mechanistic studies in cell models:

  • Examine TIMELESS upregulation in response to SARS-CoV-2 spike protein exposure

  • Use RNA-seq after TIMELESS knockdown to identify downstream pathways

Key Research Findings:
Functional analyses using gene ontology terms and the Kyoto Encyclopedia of Genes and Genomes pathway have suggested that TIMELESS contributes to the production of antiphospholipid antibodies and thrombosis in both COVID-19 and antiphospholipid syndrome (APS) patients. GSK3B has been found to be associated with TIMELESS in this context .

These approaches can help elucidate the molecular mechanisms connecting circadian clock proteins like TIMELESS to immune dysregulation and coagulation disorders in COVID-19, potentially leading to novel therapeutic strategies targeting autophagy pathways .

What methodologies are recommended for studying TIMELESS in cancer resistance mechanisms?

TIMELESS has been implicated in cancer therapy resistance, particularly in nasopharyngeal carcinoma, warranting specific methodological approaches when using biotin-conjugated antibodies:

Tissue Microarray Analysis:

• Optimize antigen retrieval for FFPE cancer tissues (TE buffer pH 9.0)

• Establish scoring system for nuclear TIMELESS expression

• Correlate with patient treatment response and survival data

Functional Studies in Cancer Cell Lines:

• Therapy resistance models:

  • Generate cisplatin-resistant cell lines through gradual exposure

  • Compare TIMELESS expression before and after resistance development

  • Use biotin-conjugated antibodies for quantitative flow cytometry analysis

• Pathway analysis:

  • Co-stain for TIMELESS and β-catenin to assess Wnt pathway activation

  • Evaluate epithelial-mesenchymal transition (EMT) markers in relation to TIMELESS expression

In vivo xenograft approaches:

• Use antibodies to monitor TIMELESS expression in excised tumors

• Compare treatment-responsive vs. treatment-resistant regions within tumors

• Correlate with markers of proliferation and stemness

Recent research has demonstrated that TIMELESS confers cisplatin resistance in nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway and promoting epithelial-mesenchymal transition , suggesting a potential therapeutic target for overcoming chemoresistance.

How can researchers optimize experiments studying TIMELESS function in DNA damage response using biotin-conjugated antibodies?

TIMELESS plays a crucial role in DNA damage response, making it an important target for cancer and genomic stability research. Optimization of biotin-conjugated antibody usage in this context includes:

Laser Microirradiation Studies:

• Pre-sensitization: Treat cells with BrdU (10 μM) for 24 hours prior to laser microirradiation

• Real-time recruitment: Use streptavidin-fluorophore conjugates for live-cell imaging of TIMELESS recruitment

• Co-recruitment analysis: Study temporal relationship between TIMELESS and other repair factors (PARP1, RAD51)

DNA Damage Response Analysis:

• IR treatment paradigm: Expose cells to 2-10 Gy ionizing radiation and monitor TIMELESS localization at 0.5, 2, 6, and 24 hours post-irradiation

• Replication stress inducers: Compare responses to different stressors (hydroxyurea, aphidicolin, UV)

• Checkpoint analysis: Correlate with CHK1 phosphorylation status and cell cycle progression

Advanced microscopy approaches:

• FRAP analysis: Study mobility of TIMELESS at damage sites using photobleaching techniques

• Super-resolution microscopy: Resolve sub-nuclear structures using techniques like STORM or PALM with biotin-conjugated antibodies and fluorescent streptavidin

Research findings indicate that in response to double-strand breaks, TIMELESS accumulates at DNA damage sites and promotes homologous recombination repair through interaction with PARP1 . This provides a mechanistic framework for investigating how TIMELESS coordinates the cellular response to genotoxic stress.

What strategies can resolve high background when using biotin-conjugated TIMELESS antibodies in tissues with endogenous biotin?

Working with biotin-conjugated antibodies in tissues with high endogenous biotin levels presents specific challenges that require targeted solutions:

Pre-analytical Approaches:

• Avidin/Biotin blocking kit: Apply sequentially before antibody incubation:

  • Incubate with avidin solution (15 min)

  • Wash briefly

  • Apply biotin solution (15 min)

  • Wash thoroughly

• Tissue-specific considerations:

  • For liver, kidney, and brain tissues (high endogenous biotin): Extend blocking time to 30 minutes

  • For spleen and lymphoid tissues: Include additional 10% normal serum in blocking solution

Analytical Optimizations:

• Antibody dilution: Use higher dilutions (1:200-1:500) to reduce non-specific binding

• Streptavidin-conjugate selection: Try different formats (HRP vs. fluorophore-conjugated)

• Incubation conditions: Perform at 4°C overnight rather than room temperature

Alternative Detection Methods:

• Tyramide signal amplification: Lower antibody concentration while maintaining sensitivity

• Polymer-based detection systems: Combine with brief biotin-conjugated antibody exposure

Validation Approach:
Include a biotinylated control antibody of the same isotype but irrelevant specificity to distinguish between specific staining and endogenous biotin background .

These techniques are particularly important when examining TIMELESS expression in tissues like brain, heart, and liver where endogenous biotin can significantly impact signal-to-noise ratios .

How can researchers optimize double immunofluorescence protocols using biotin-conjugated TIMELESS antibodies and other non-biotinylated antibodies?

Combining biotin-conjugated TIMELESS antibodies with other detection systems requires specific optimization strategies:

Sequential Protocol Optimization:

• Order of application:

  • First detect the non-biotinylated antibody with a direct fluorophore-conjugated secondary

  • Block any open binding sites with excess secondary antibody

  • Then apply biotin-conjugated TIMELESS antibody followed by streptavidin-fluorophore

• Concentration balancing:

  • Titrate both antibodies individually first

  • Use the biotin-conjugated TIMELESS antibody at a higher dilution (1:200-1:500)

  • Adjust streptavidin-fluorophore concentration (typically 1:100-1:500)

Cross-reactivity Prevention:

• Secondary antibody selection: Choose secondaries raised in different host species than the primary antibodies

• Absorption controls: Pre-absorb secondaries against irrelevant species IgG

• Blocking optimization: Include both serum and IgG from species of all secondaries

Advanced Options:

• Zenon labeling technology: Consider direct labeling of the non-biotinylated primary

• Sequential image acquisition: Acquire first fluorochrome, photobleach, then detect biotin

• Spectral imaging: Use systems with spectral unmixing capabilities to separate overlapping signals

These approaches enable reliable co-localization studies of TIMELESS with partners like TIPIN or replication fork components while minimizing artifacts .

What are the most effective approaches for quantifying TIMELESS protein levels using biotin-conjugated antibodies across different experimental platforms?

Accurate quantification of TIMELESS protein levels requires platform-specific optimization when using biotin-conjugated antibodies:

Western Blot Quantification:

• Standard curve approach: Include recombinant TIMELESS protein at known concentrations

• Normalization strategy: Use nuclear-specific loading controls (Lamin B1 or Histone H3)

• Signal development: Use chemiluminescent substrates with extended linear range

• Analysis software: Apply background subtraction and lane normalization

Flow Cytometry Quantification:

• Calibration beads: Use anti-mouse IgG beads with known antibody binding capacity

• Signal amplification: Multi-layer approach with biotin-streptavidin systems

• Controls: Include fluorescence minus one (FMO) and isotype controls

• Gating strategy: Apply consistent nuclear gating based on DNA content

Tissue Analysis Quantification:

• Digital pathology approach: Use whole slide imaging with automated analysis

• Machine learning algorithms: Train on known positive and negative controls

• Multiplexed normalization: Include nuclear stain and housekeeping protein

• Spatial analysis: Distinguish nuclear vs. cytoplasmic signal intensity

Standardization Across Platforms:
Implement relative quantification using reference cell lines with known TIMELESS expression levels (e.g., HeLa cells) processed in parallel with experimental samples .

These quantification strategies enable reliable comparison of TIMELESS protein levels across experimental conditions, cell types, and disease states, essential for understanding its role in normal physiology and pathological conditions .