POLE4 Antibody, Biotin conjugated

What is POLE4 and what is its biological significance in DNA replication studies?

POLE4, also known as DNA polymerase epsilon subunit 4 (p12 subunit), is an essential component of the DNA polymerase epsilon complex that plays a critical role in chromosomal DNA replication and repair mechanisms . The protein consists of 117 amino acids with a calculated molecular weight of approximately 12 kDa, although it typically appears at around 17 kDa in Western blot analyses due to post-translational modifications .

POLE4 functions as an accessory component within the larger DNA polymerase epsilon complex, which is responsible for the synthesis of the leading strand during DNA replication . Research has demonstrated that POLE4 is critical for maintaining genomic stability, as dysregulation of this protein has been implicated in various diseases, including cancer, where mutations can lead to genomic instability and tumor development .

What experimental applications are biotin-conjugated POLE4 antibodies validated for?

Biotin-conjugated POLE4 antibodies have been primarily validated for the following applications:

When designing experiments, researchers should note that while ELISA applications are consistently validated across multiple products, other applications may require additional optimization or specific product selection based on the experimental needs .

How should POLE4 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of POLE4 antibodies, particularly biotin-conjugated versions, is critical for maintaining their activity and specificity:

• Storage temperature: Store at -20°C for long-term stability. Some manufacturers suggest that the antibody is stable for one year after shipment when properly stored .

• Buffer composition: Most POLE4 antibodies are supplied in buffers containing:

  • PBS (pH 7.4)

  • 50% Glycerol

  • Preservatives such as 0.03% Proclin 300 or sodium azide (0.02%)

• Aliquoting recommendations: For -20°C storage, aliquoting is generally unnecessary for small volumes (20μL), but larger volumes should be divided to avoid repeated freeze-thaw cycles .

• Thawing protocol: Thaw on ice and keep cold during handling to preserve antibody integrity.

• Working dilution preparation: Prepare working dilutions on the day of the experiment for optimal results .

How can researchers validate the specificity of biotin-conjugated POLE4 antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For biotin-conjugated POLE4 antibodies, consider these comprehensive validation strategies:

• Positive control selection: Use cell lines known to express POLE4, such as HeLa, 293T, HepG2, MCF-7, and HL-60, which have been confirmed as positive samples for POLE4 expression .

• Knockdown/knockout verification: Compare antibody reactivity in wild-type cells versus those with POLE4 knockdown or knockout to confirm signal specificity.

• Recombinant protein competition: Pre-incubate the antibody with recombinant POLE4 protein (such as the immunogen sequence AA 8-117 or 1-117) prior to application to determine if the specific signal is abolished .

• Cross-reactivity assessment: Although most biotin-conjugated POLE4 antibodies are specifically reactive with human samples, some show cross-reactivity with mouse and rat samples. Researchers should verify the species reactivity relevant to their experiments .

• Western blot verification: Confirm the molecular weight of the detected protein matches the expected size for POLE4 (calculated MW: 12kDa, observed MW: 17kDa) .

What technical considerations are important when using biotin-conjugated antibodies in tissues with endogenous biotin?

When working with biotin-conjugated POLE4 antibodies in tissue samples, researchers must address the potential for endogenous biotin interference:

• Endogenous biotin blocking: Prior to applying biotin-conjugated antibodies, block endogenous biotin using:

  • Avidin/biotin blocking kit

  • Streptavidin/biotin blocking solutions

  • Pre-incubation with unconjugated streptavidin

• Tissue-specific considerations: Certain tissues (liver, kidney, brain) have higher endogenous biotin levels that require more stringent blocking protocols.

• Control experiments: Always include a negative control omitting the primary antibody but including all detection reagents to assess background signal from endogenous biotin.

• Alternative detection strategies: For tissues with particularly high endogenous biotin, consider using POLE4 antibodies with alternative conjugates (e.g., HRP or FITC) that are also commercially available .

• Signal amplification method selection: When using biotin-conjugated antibodies, carefully select compatible detection systems that minimize background from endogenous biotin while maximizing specific signal detection.

How does the amino acid sequence targeted by different POLE4 antibodies affect their experimental utility?

Different commercial POLE4 antibodies target various epitopes within the protein, which can significantly impact their experimental applications:

The choice of epitope region affects:

• Isoform detection capability: Antibodies targeting regions common to all isoforms will detect multiple variants, while those targeting unique regions may be isoform-specific.

• Protein interaction studies: Antibodies targeting interaction domains may interfere with protein-protein interactions, potentially limiting their use in co-immunoprecipitation studies.

• Post-translational modification detection: Consider whether the epitope contains known modification sites that could affect antibody binding.

• Structural accessibility: Some epitopes may be more accessible in the native protein conformation, making certain antibodies better suited for applications requiring detection of non-denatured proteins .

What methodological approaches can resolve discrepancies between calculated and observed molecular weights of POLE4?

Researchers frequently observe POLE4 at approximately 17 kDa on Western blots despite its calculated molecular weight of 12 kDa . To investigate and resolve this discrepancy:

• Post-translational modification analysis:

  • Treat samples with phosphatase inhibitors versus without to determine if phosphorylation contributes to the increased mass

  • Use specific glycosidases to evaluate glycosylation contributions

• SDS-PAGE optimization:

  • Run gradient gels (4-20%) to improve resolution of lower molecular weight proteins

  • Use specialized low-molecular-weight markers for more accurate size determination

• Sample preparation variables:

  • Compare different lysis buffers to ensure complete protein denaturation

  • Test various reducing conditions to fully break disulfide bonds

• Protein sequence analysis:

  • Analyze the full sequence (MAAAAAAGSGTPREEEGPAGEAAASQPQAPTSVPGARLSRLPLARVKALVKADPDVTLAGQEAIFILARAAELFVETIAKDAYCCAQQGKRKTLQRRDLDNAIEAVDEFAFLEGTLD) for regions likely to affect electrophoretic mobility

• Isoform identification:

  • Use mass spectrometry to definitively identify the protein and any modifications

  • Compare migration patterns across multiple cell lines to identify cell-specific variations

How can biotin-conjugated POLE4 antibodies be integrated into multicolor immunostaining protocols?

Biotin-conjugated POLE4 antibodies offer significant advantages in multicolor immunostaining due to their versatility with various streptavidin-conjugated detection systems:

• Sequential detection strategy:

  • Apply unconjugated primary antibodies first

  • Add species-specific secondary antibodies with direct fluorophore conjugates

  • Apply biotin-conjugated POLE4 antibody

  • Detect with streptavidin conjugated to a spectrally distinct fluorophore

• Multiplexing considerations:

  • Select fluorophores with minimal spectral overlap

  • Use proper absorption/emission filters to prevent bleed-through

  • Include appropriate single-stained controls for accurate compensation

• Signal amplification options:

  • Utilize tyramide signal amplification (TSA) with biotin-conjugated antibodies for detecting low-abundance POLE4

  • Employ streptavidin-conjugated quantum dots for enhanced photostability in long imaging sessions

• Subcellular localization studies:

  • Pair POLE4 detection with nuclear markers to confirm its expected nuclear localization

  • Consider co-staining with other DNA replication complex components to study protein interactions

What approaches can researchers use to study POLE4's role in DNA repair mechanisms using biotin-conjugated antibodies?

To investigate POLE4's involvement in DNA repair pathways using biotin-conjugated antibodies:

• Damage-induced localization studies:

  • Induce DNA damage using UV irradiation, radiomimetic drugs, or site-specific nucleases

  • Track POLE4 recruitment to damage sites using biotin-conjugated antibodies

  • Quantify co-localization with known DNA damage response proteins

• Chromatin association dynamics:

  • Perform chromatin fractionation followed by Western blotting

  • Compare POLE4 chromatin association before and after DNA damage

  • Use biotin-conjugated antibodies for detection in these biochemical assays

• Functional interaction studies:

  • Deplete other DNA polymerase epsilon complex components and assess POLE4 localization

  • Examine how POLE4 depletion affects recruitment of other repair factors

  • Utilize proximity ligation assays with biotin-conjugated POLE4 antibodies to detect protein-protein interactions in situ

• Cell cycle-dependent regulation:

  • Synchronize cells at different cell cycle phases

  • Assess POLE4 expression, localization, and protein interactions throughout the cell cycle

  • Correlate findings with DNA repair efficiency measurements

What experimental controls are essential when using biotin-conjugated POLE4 antibodies in complex tissue samples?

When applying biotin-conjugated POLE4 antibodies to tissue samples, implement these critical controls:

• Absorption controls:

  • Pre-incubate antibody with recombinant POLE4 protein containing the target epitope (AA 8-117)

  • Apply this pre-absorbed antibody to parallel tissue sections to confirm signal specificity

• Tissue-specific controls:

  • Include tissues known to be negative for POLE4 expression

  • Use tissues from POLE4 knockout models when available

  • Compare with tissues known to have high POLE4 expression

• Detection system controls:

  • Include streptavidin-only controls (omitting primary antibody)

  • Use non-biotinylated primary antibodies with the same detection system

  • Apply detection reagents to serial sections pre-blocked with unlabeled avidin/streptavidin

• Fixation method comparison:

  • Compare results between different fixation methods (PFA, methanol, acetone)

  • Evaluate epitope retrieval methods for optimal signal-to-noise ratio

• Cross-reactivity assessment:

  • Test antibody on tissue from different species to confirm specificity claims

  • Include appropriate blocking reagents to minimize non-specific binding

How can researchers troubleshoot weak or inconsistent signals when using biotin-conjugated POLE4 antibodies?

When facing detection challenges with biotin-conjugated POLE4 antibodies, consider these systematic troubleshooting approaches:

• Antibody concentration optimization:

  • Test a range of dilutions beyond the manufacturer's recommended range (e.g., 1:250 - 1:2,000)

  • Consider that different applications may require significantly different concentrations

• Sample preparation refinement:

  • Evaluate multiple lysis buffers for protein extraction efficiency

  • Compare different antigen retrieval methods for fixed samples

  • Test fresh versus frozen samples for signal integrity

• Detection system enhancement:

  • Implement amplification steps using tyramide signal amplification

  • Compare different streptavidin conjugates for optimal sensitivity

  • Extend incubation times for both primary antibody and detection reagents

• Buffer composition modification:

  • Adjust blocking reagents to reduce background while preserving specific signal

  • Test different detergents and concentrations in wash buffers

  • Consider adding protein stabilizers to dilution buffers

• Positive control inclusion:

  • Always run recommended positive control cell lines (HeLa, 293T, HepG2, MCF-7, HL-60)

  • Include recombinant POLE4 protein as a definitive positive control

What methodological approaches can integrate POLE4 detection with studies of genomic instability?

To investigate POLE4's relationship with genomic instability mechanisms:

• Combined immunofluorescence/FISH protocols:

  • Detect POLE4 using biotin-conjugated antibodies

  • Perform fluorescence in situ hybridization for common fragile sites or specific chromosome regions

  • Quantify co-localization between POLE4 and sites of genomic instability

• Replication stress analysis:

  • Induce replication stress using hydroxyurea or aphidicolin

  • Monitor POLE4 expression and localization changes

  • Correlate with markers of replication fork stalling (γH2AX, RPA foci)

• Cell synchronization strategies:

  • Synchronize cells at different cell cycle phases

  • Assess POLE4 levels and localization throughout the cycle

  • Connect findings to replication timing and error rates

• Mutation spectrum analysis:

  • Manipulate POLE4 expression in cell models

  • Perform whole-genome sequencing to assess mutation patterns

  • Correlate mutation signatures with POLE4 function

This analytical approach provides mechanistic insights into how POLE4 dysfunction contributes to the genomic instability observed in cancer contexts .

How do different host species and clonality affect POLE4 antibody performance in various applications?

The host species and clonality characteristics significantly impact experimental outcomes when using POLE4 antibodies:

Key performance considerations:

• Host species selection:

  • Rabbit-derived antibodies generally show high affinity and are versatile across applications

  • Mouse-derived antibodies may present challenges when studying mouse tissues due to high background

• Clonality impact:

  • Polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals

  • Batch-to-batch variability can be greater with polyclonal antibodies

  • Different lots should be validated for consistency in critical experiments

• Cross-reactivity considerations:

  • Some POLE4 antibodies show cross-reactivity with multiple species (human, mouse, rat)

  • Others are human-specific and should not be used for cross-species studies

  • Cross-reactivity should be experimentally verified rather than assumed

• Application-specific performance:

  • For challenging applications like chromatin immunoprecipitation, antibody selection is particularly critical

  • For multi-color imaging, consider how the host species affects secondary antibody selection to avoid cross-reactivity

By carefully considering these factors, researchers can select the optimal POLE4 antibody configuration for their specific experimental needs.