POLE4 Antibody, FITC conjugated

What is POLE4 and what biological functions does it serve?

POLE4 (DNA polymerase epsilon subunit 4) is a histone-fold protein that plays a critical role in DNA replication and repair mechanisms. It interacts with other histone-fold proteins to bind DNA in a sequence-independent manner, forming dimers that combine within larger enzymatic complexes for DNA transcription, replication, and packaging . The protein functions primarily by allowing polymerase epsilon to effectively carry out its replication and repair functions within the cell .

POLE4 has a molecular weight of approximately 12,209 Da and is also known by several synonyms including DNA polymerase II subunit 4 and DNA polymerase epsilon subunit p12 . According to NCBI annotation, POLE4 is considered an accessory subunit to the DNA polymerase epsilon complex, which is essential for faithful DNA replication in eukaryotic cells . The protein's involvement in fundamental cellular processes makes it a valuable target for research investigating DNA replication dynamics, genome stability, and potential implications in diseases characterized by replication errors.

What applications is POLE4 Antibody, FITC conjugated suitable for?

POLE4 Antibody, FITC conjugated is primarily validated for use in ELISA (Enzyme-Linked Immunosorbent Assay) applications . The FITC conjugation enables fluorescent detection with excitation maximum at approximately 495 nm and emission maximum at 525 nm, making it suitable for various fluorescence-based detection methods .

While ELISA is the validated application, FITC-conjugated antibodies are generally applicable for immunohistochemistry and immunofluorescence studies utilizing flow cytometry . The antibody could potentially be used in these additional applications, though researchers would need to validate its performance for their specific experimental systems. The FITC conjugation allows for direct detection without the need for secondary antibodies, simplifying experimental workflows and potentially reducing background signals in certain experimental setups.

What are the technical specifications of POLE4 Antibody, FITC conjugated?

The POLE4 Antibody, FITC conjugated is available as a polyclonal antibody raised in rabbit against recombinant Human DNA polymerase epsilon subunit 4 protein (specifically amino acids 8-117) . It demonstrates reactivity with human POLE4 protein and is of IgG isotype . Technical specifications include:

The antibody is purified using Protein G affinity chromatography to a purity level of >95%, ensuring high specificity for research applications .

How should POLE4 Antibody, FITC conjugated be stored to maintain its activity?

Proper storage is critical for maintaining antibody activity and fluorescence intensity. POLE4 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can lead to antibody degradation and loss of FITC fluorescence intensity .

For working solutions, it is advisable to prepare small aliquots for single use to minimize repeated freeze-thaw cycles. The storage buffer (containing 50% Glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as preservative) helps maintain antibody stability during storage . When handling the antibody, it should be protected from prolonged exposure to light to prevent photobleaching of the FITC fluorophore, which could result in diminished fluorescence signal intensity during experimental applications.

How does the FITC conjugation process affect the binding properties of the POLE4 antibody?

The FITC conjugation to POLE4 antibody involves a chemical reaction between fluorescein isothiocyanate and free amino groups (primarily lysine residues) of the antibody protein . This conjugation process can potentially affect the antibody's binding properties depending on the location of the modified amino groups. If lysine residues are present in or near the antigen-binding site (paratope), conjugation could potentially impact antigen recognition and binding affinity.

The fluorescein/protein (F/P) ratio is a critical parameter that affects both the fluorescence intensity and the antibody's functionality. For IgG antibodies, optimal F/P ratios typically range between 2-4 molecules of FITC per antibody molecule . Lower ratios may result in insufficient fluorescence signal, while higher ratios can cause excessive conjugation that may interfere with antigen binding or lead to fluorescence quenching. For POLE4 antibody specifically, researchers should validate the effect of FITC conjugation on antigen binding by comparing the performance of conjugated and unconjugated antibodies in parallel experiments to ensure that sensitivity and specificity remain adequate for the intended application.

What methodological considerations are important when using FITC-conjugated antibodies in fluorescence microscopy or flow cytometry?

When using POLE4 Antibody, FITC conjugated for fluorescence microscopy or flow cytometry, several methodological considerations are essential for optimal results:

• Spectral Characteristics: FITC has an absorption maximum at 495 nm and emission maximum at 525 nm, requiring appropriate filter sets for detection . Researchers should ensure their instrumentation has compatible excitation sources and emission filters.

• Photobleaching Mitigation: FITC is susceptible to photobleaching, requiring measures such as anti-fade mounting media for microscopy, minimal exposure to excitation light, and storage of samples protected from light .

• pH Sensitivity: FITC fluorescence is pH-dependent, with optimal fluorescence at slightly alkaline conditions (pH 8-9). Researchers should maintain consistent pH during experiments to ensure reproducible signal intensity .

• Autofluorescence Control: Cellular components can exhibit autofluorescence in the same spectral range as FITC. Proper controls including unstained cells and isotype controls are essential to distinguish specific from non-specific signals .

• Signal Amplification: For low-abundance targets like certain POLE4 populations, signal amplification methods may be necessary, such as biotin-streptavidin systems or tyramide signal amplification, though these would require additional validation with the POLE4 antibody .

Careful optimization of antibody concentration, incubation conditions, and washing steps is essential for maximizing specific signal while minimizing background fluorescence.

What experimental controls should be implemented when using POLE4 Antibody, FITC conjugated?

Implementing appropriate experimental controls is essential for accurate interpretation of results when using POLE4 Antibody, FITC conjugated:

• Isotype Control: A FITC-conjugated rabbit IgG antibody (non-specific) should be used at the same concentration as the POLE4 antibody to assess non-specific binding and establish background fluorescence levels .

• Blocking Controls: Pre-incubation of the antibody with recombinant POLE4 protein (particularly the immunogen sequence 8-117AA) should abolish specific staining, confirming antibody specificity .

• Positive Controls: Cell lines or tissues known to express POLE4 should be included to verify antibody performance. Given POLE4's role in DNA replication, proliferating cells would be appropriate positive controls .

• Negative Controls: Cell lines with POLE4 knockdown/knockout or tissues known not to express POLE4 should demonstrate minimal signal.

• Secondary Antibody Control: Though not directly applicable for FITC-conjugated antibodies, this control helps assess potential non-specific binding of detection systems in multi-step staining protocols.

• Autofluorescence Control: Unstained samples should be analyzed to determine baseline cellular autofluorescence, particularly important when working with tissues containing collagen or lipofuscin.

• Absorption Controls: When multiplexing with other fluorophores, single-stained controls help assess and correct for spectral overlap.

What is the recommended protocol for using POLE4 Antibody, FITC conjugated in ELISA?

While specific protocols for POLE4 Antibody, FITC conjugated in ELISA are not provided in the search results, a general protocol for FITC-conjugated antibodies in ELISA can be adapted as follows:

• Plate Coating: Coat a high-binding ELISA plate with capture antigen (recombinant POLE4 protein) or for sandwich ELISA, coat with anti-POLE4 capture antibody at 1-10 μg/ml in coating buffer (typically carbonate/bicarbonate buffer, pH 9.6). Incubate overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA or non-fat dry milk in PBS for 1-2 hours at room temperature.

• Sample Addition: For direct ELISA, add test samples containing POLE4. For sandwich ELISA, add samples containing the target antigen. Incubate for 1-2 hours at room temperature.

• Primary Antibody: Add POLE4 Antibody, FITC conjugated at an optimized concentration (typical starting range 1-10 μg/ml) diluted in blocking buffer. Incubate for 1-2 hours at room temperature, protected from light.

• Washing: Wash thoroughly (4-6 times) with PBS containing 0.05% Tween-20 (PBST) to remove unbound antibody.

• Detection: For FITC detection in ELISA, either:

  • Use a fluorescence microplate reader with appropriate excitation (495 nm) and emission (525 nm) settings

  • Or, use an anti-FITC antibody conjugated with HRP, followed by addition of a chromogenic substrate like TMB, and measure absorbance

• Data Analysis: Generate a standard curve using known concentrations of recombinant POLE4 protein to quantify samples.

Optimization is crucial for each step, including antibody concentration, incubation times and temperatures, and washing stringency to achieve optimal signal-to-noise ratio .

How can POLE4 Antibody, FITC conjugated be used for intracellular staining in flow cytometry?

Using POLE4 Antibody, FITC conjugated for intracellular staining in flow cytometry requires specific methodological considerations due to POLE4's intracellular localization as a nuclear protein involved in DNA replication:

• Cell Preparation:

  • Harvest cells and wash in PBS

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Wash fixed cells with PBS containing 1% BSA

• Permeabilization:

  • Permeabilize cells using 0.1-0.5% saponin, 0.1-0.5% Triton X-100, or commercially available permeabilization buffers

  • More stringent permeabilization may be required for nuclear antigens like POLE4

  • For optimal nuclear protein detection, methanol permeabilization (100% ice-cold methanol for 10 minutes) can be considered

• Blocking:

  • Block with 5-10% normal serum (not from the same species as the primary antibody) in permeabilization buffer for 30 minutes

• Antibody Staining:

  • Dilute POLE4 Antibody, FITC conjugated in permeabilization buffer containing blocking serum

  • Initial titration experiments should be performed to determine optimal antibody concentration

  • Incubate for 30-60 minutes at room temperature or 4°C, protected from light

• Washing:

  • Wash 2-3 times with permeabilization buffer

  • Final wash with PBS/BSA

• Flow Cytometry Analysis:

  • Resuspend cells in appropriate buffer for flow cytometric analysis

  • Use appropriate instrument settings for FITC detection (excitation ~488 nm, emission ~525 nm)

  • Include proper controls as described in section 2.4

Since POLE4 expression may vary during the cell cycle (being potentially higher during S phase due to its role in DNA replication), co-staining with cell cycle markers might provide additional biological insights .

What sample preparation techniques are recommended for detecting POLE4 in various cellular compartments?

POLE4 is primarily localized in the nucleus as part of the DNA replication machinery, requiring specific sample preparation techniques for optimal detection in different experimental setups:

• Cell Lysis for Biochemical Analysis:

  • For nuclear protein extraction, specialized nuclear extraction protocols are recommended

  • Use a cellular fractionation approach to separate nuclear from cytoplasmic components:

    • Initial lysis with non-ionic detergent (e.g., 0.1% NP-40) to release cytoplasmic contents

    • Nuclear pellet extraction with higher detergent concentration and/or sonication

  • Protease inhibitors should be included in all buffers to prevent degradation

  • Phosphatase inhibitors may be relevant if studying POLE4 phosphorylation status

• Fixation for Microscopy:

  • Cross-linking fixatives (4% paraformaldehyde, 10-15 minutes) preserve protein localization

  • For better nuclear antigen access, consider additional permeabilization with 0.5% Triton X-100

  • Methanol fixation (-20°C, 10 minutes) may provide better access to nuclear antigens

• Tissue Preparation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues may require antigen retrieval methods

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Fresh frozen tissues may provide better antigen preservation but require careful handling

• Chromatin Immunoprecipitation (ChIP):

  • If studying POLE4 association with DNA, optimized ChIP protocols using cross-linking reagents like formaldehyde

  • Sonication parameters should be optimized for appropriate chromatin fragmentation

  • Specialized buffers for nuclear protein extraction and DNA-protein complex isolation

When investigating POLE4 in relation to the cell cycle, synchronization techniques (e.g., double thymidine block for S-phase enrichment) may provide more consistent results due to POLE4's function in DNA replication .

How can I troubleshoot weak or no signal when using POLE4 Antibody, FITC conjugated?

When encountering weak or no signal with POLE4 Antibody, FITC conjugated, systematic troubleshooting can help identify and resolve the issue:

• Antibody Activity Issues:

  • Verify antibody storage conditions (-20°C to -80°C, protected from light)

  • Check for excessive freeze-thaw cycles which can diminish activity

  • Assess fluorophore integrity by measuring FITC absorbance at 495 nm

  • Consider fluorescein/protein (F/P) ratio determination to ensure adequate labeling

• Protocol Optimization:

  • Increase antibody concentration in a titration experiment

  • Extend incubation time or adjust temperature

  • Modify blocking conditions to reduce background while preserving specific signal

  • For intracellular targets like POLE4, ensure adequate permeabilization for antibody access

• Sample-Related Issues:

  • Confirm POLE4 expression in your sample type (consider positive control cells)

  • Assess protein integrity in lysates using total protein stains

  • For fixed samples, optimize fixation conditions and include antigen retrieval steps

  • Consider cell cycle stage, as POLE4 expression may vary during replication phases

• Detection System Problems:

  • Verify instrument settings (excitation ~495 nm, emission ~525 nm for FITC)

  • Check filter sets for compatibility with FITC spectral characteristics

  • Assess detector sensitivity and gain settings

  • For microscopy, evaluate objective lens quality and light source intensity

• Technical Considerations:

  • Reduce exposure to light during all steps

  • Use freshly prepared buffers at optimal pH (FITC fluorescence is pH-sensitive)

  • Include antifade reagents to minimize photobleaching

  • Test for potential interfering substances in buffers or samples

A systematic approach comparing experimental samples with appropriate controls can help isolate the source of signal problems and guide appropriate troubleshooting strategies .

How can I validate the specificity of POLE4 Antibody, FITC conjugated in my experimental system?

Validating the specificity of POLE4 Antibody, FITC conjugated is crucial for ensuring reliable research data. Multiple complementary approaches should be employed:

• Competitive Inhibition:

  • Pre-incubate the antibody with excess recombinant POLE4 protein (specifically the immunogen sequence 8-117AA)

  • Compare staining patterns with and without competitive inhibition

  • Specific signals should be significantly reduced or eliminated with pre-absorption

• Genetic Validation:

  • Use POLE4 knockout/knockdown cells (CRISPR-Cas9, siRNA, or shRNA approaches)

  • Compare signal between wild-type and POLE4-depleted samples

  • Specific antibodies should show reduced signal proportional to knockdown efficiency

• Orthogonal Detection:

  • Compare staining patterns with alternative POLE4 antibodies targeting different epitopes

  • Concordant results across different antibodies increase confidence in specificity

  • Use alternative detection methods (western blot, immunoprecipitation) to confirm target specificity

• Biological Validation:

  • Test correlation with expected biological behaviors (e.g., nuclear localization, cell cycle-dependent expression)

  • Confirm colocalization with known POLE4 interaction partners in the DNA polymerase epsilon complex

  • Verify patterns consistent with POLE4's role in DNA replication

• Technical Controls:

  • Include isotype controls at equivalent concentrations to assess non-specific binding

  • Test reactivity in cells/tissues known to express or lack POLE4

  • Evaluate signal correlation with independent measures of POLE4 expression (e.g., mRNA levels)

Documentation of these validation steps provides crucial evidence of antibody specificity and strengthens the reliability of experimental findings using POLE4 Antibody, FITC conjugated .

What is the significance of the fluorescein/protein (F/P) ratio in experimental outcomes?

The fluorescein/protein (F/P) ratio significantly impacts experimental outcomes when using POLE4 Antibody, FITC conjugated, affecting both signal quality and antibody functionality:

For critical applications, researchers may need to determine the F/P ratio of their POLE4 Antibody, FITC conjugated using spectrophotometric methods and optimize experimental conditions accordingly to ensure consistent, reliable results .

What future research directions might benefit from POLE4 Antibody, FITC conjugated applications?

POLE4 Antibody, FITC conjugated represents an important tool for advancing research in several promising directions related to DNA replication, genome stability, and disease mechanisms:

• Cell Cycle Regulation Studies: The involvement of POLE4 in DNA replication makes this antibody valuable for investigating cell cycle dynamics, particularly during S-phase progression. Researchers can use FITC-conjugated POLE4 antibody for real-time tracking of POLE4 localization and abundance throughout the cell cycle, potentially revealing regulatory mechanisms controlling DNA replication timing and fidelity .

• Cancer Research Applications: DNA replication errors contribute to genomic instability in cancer. POLE4 Antibody, FITC conjugated could help investigate the role of DNA polymerase epsilon complex dysregulation in tumorigenesis, potentially identifying novel diagnostic markers or therapeutic targets. The antibody may be particularly valuable for analyzing POLE4 expression patterns in cancer tissues using flow cytometry or immunohistochemistry approaches .

• DNA Damage Response Pathways: Given POLE4's role in DNA repair, the antibody could facilitate studies examining how cells respond to genotoxic stress. Researchers could track POLE4 recruitment to sites of DNA damage using live-cell imaging with the FITC-conjugated antibody, potentially revealing new insights into repair pathway choice and efficiency .

• Protein-Protein Interaction Networks: POLE4 functions within larger protein complexes for DNA processing. The FITC-conjugated antibody could support co-localization studies, fluorescence resonance energy transfer (FRET) experiments, or proximity ligation assays to map the dynamic interactions between POLE4 and other replication factors under various cellular conditions .

• Drug Development Screening: Compounds affecting DNA replication might alter POLE4 expression, localization, or complex formation. The antibody could serve in high-throughput screening assays to identify molecules that modulate POLE4 activity, potentially leading to new classes of antiviral or anticancer therapeutics targeting DNA replication machinery .