RFC4 Antibody, FITC conjugated

What is RFC4 and what is its role in cellular processes?

RFC4 (Replication Factor C Subunit 4) is a 37 kDa protein that functions as a critical component of DNA replication machinery. It serves as a subunit of Activator 1, which works in conjunction with proliferating cell nuclear antigen (PCNA) to enable the elongation of primed DNA templates by DNA polymerase delta and epsilon . RFC4 is specifically involved in the elongation of multiprimed DNA templates, making it essential for efficient and accurate DNA replication . This protein is primarily active in nuclear signaling pathways and epigenetic regulation processes . As part of the replication factor C complex, it helps load PCNA onto DNA, which then serves as a processivity factor for DNA polymerases during replication.

What are the spectral properties of FITC conjugation and how do they impact experimental design?

FITC (Fluorescein Isothiocyanate) conjugated to RFC4 antibodies has specific spectral properties that researchers must consider in experimental design. The FITC fluorophore has an excitation maximum at 495 nm and an emission maximum at 519 nm . These spectral characteristics make FITC-conjugated antibodies compatible with standard fluorescence microscopy filter sets and flow cytometry instruments equipped with 488 nm lasers.

When designing multiplex experiments, researchers should account for potential spectral overlap with other fluorophores. The relatively broad emission spectrum of FITC may overlap with fluorophores such as GFP or PE, which should be considered when selecting complementary fluorophores for multi-color experiments. Additionally, FITC is susceptible to photobleaching and pH sensitivity, which necessitates proper sample handling and buffer selection to maintain signal integrity throughout the experiment.

How does the molecular F/P ratio affect antibody performance in RFC4-FITC conjugates?

The fluorescein/protein (F/P) ratio is a critical parameter affecting the performance of FITC-conjugated RFC4 antibodies. Optimal labeling is achieved when the F/P ratio is balanced—neither too low (resulting in weak signal) nor too high (causing quenching effects and increased non-specific binding) .

Research indicates that maximal labeling efficiency is reached within 30-60 minutes at room temperature, pH 9.5, and an initial protein concentration of 25 mg/ml . The separation of optimally labeled antibodies from under- and over-labeled proteins can be achieved through gradient DEAE Sephadex chromatography .

The relationship between F/P ratio and antibody activity follows a bell-shaped curve:

Researchers should verify the F/P ratio of commercial RFC4-FITC antibodies before use, as this parameter significantly impacts experimental outcomes, particularly for quantitative analyses .

What are the optimal storage conditions for maintaining RFC4-FITC antibody activity?

Proper storage of RFC4-FITC conjugated antibodies is essential for maintaining their activity and fluorescence intensity over time. Based on manufacturer recommendations, RFC4-FITC antibodies should be stored at -20°C or -80°C upon receipt for long-term storage . For RFC4-FITC conjugates specifically, the optimal storage formulation typically includes 50% glycerol in 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative .

Once thawed for use, the antibody should be stored at 4°C in the dark and protected from light to prevent photobleaching of the FITC fluorophore . Repeated freeze-thaw cycles should be avoided as they can significantly degrade antibody performance . If multiple uses are anticipated, it is advisable to prepare small aliquots before freezing to minimize freeze-thaw cycles.

The following storage guidelines will help maximize antibody shelf-life:

Research has shown that properly stored FITC-conjugated antibodies maintain >90% of their activity for at least 12 months .

What dilutions and concentrations are optimal for different applications of RFC4-FITC antibodies?

The optimal dilution of RFC4-FITC antibodies varies by application and specific antibody characteristics. While manufacturers often recommend experimental determination of optimal dilutions , the following table provides general guidelines based on research experience and manufacturer data:

For RFC4-specific detection, consider that the protein has a predicted molecular weight of 40 kDa when analyzing Western blot results . Always include appropriate positive controls (such as HeLa, 293T, or K562 cell lysates for human RFC4) to validate antibody performance at your chosen dilution .

What fixation and permeabilization methods work best with RFC4-FITC antibodies?

The choice of fixation and permeabilization methods significantly impacts the performance of RFC4-FITC antibodies, particularly for applications requiring intracellular staining such as ICC and IHC. Since RFC4 is primarily a nuclear protein involved in DNA replication, proper nuclear access is essential for accurate detection.

Based on research protocols and manufacturer recommendations:

For RFC4 visualization specifically, the paraformaldehyde (4%) fixation followed by permeabilization with 0.1-0.3% Triton X-100 typically yields optimal results for immunofluorescence applications . This method provides adequate preservation of cellular architecture while allowing antibody access to the nuclear compartment where RFC4 is predominantly located.

When working with paraffin-embedded tissues, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is recommended to restore antigenicity after formalin fixation .

How can researchers quantify RFC4 expression levels using FITC-conjugated antibodies?

Quantification of RFC4 expression using FITC-conjugated antibodies can be approached through several methodologies, each appropriate for different research questions and available instrumentation.

For immunofluorescence imaging-based quantification:

• Acquire images using standardized exposure settings across all samples

• Use software like ImageJ, CellProfiler or ZEN to measure nuclear fluorescence intensity

• Apply appropriate background subtraction methods

• Normalize to nuclear area or DAPI signal intensity

• Calculate mean fluorescence intensity (MFI) across multiple cells (minimum 50-100 cells recommended)

For flow cytometry-based quantification:

• Establish appropriate gating strategy based on forward/side scatter and viability markers

• Compare RFC4-FITC signal to isotype-matched FITC-conjugated control antibodies

• Calculate median fluorescence intensity and coefficient of variation

• Consider using calibration beads to convert arbitrary fluorescence units to molecules of equivalent soluble fluorochrome (MESF)

For Western blot quantification with FITC-labeled secondary antibodies:

• Use validated loading controls (e.g., β-actin, GAPDH)

• Capture images using a fluorescence imager with appropriate filters (excitation ~495 nm, emission ~519 nm)

• Analyze band intensity using densitometry software

• Calculate relative RFC4 expression normalized to loading controls

When comparing RFC4 expression across cell cycle phases, synchronization methods should be employed and cycle stages confirmed with DNA content analysis.

What controls are essential when working with RFC4-FITC antibodies?

Implementing proper controls is critical for generating reliable and interpretable data with RFC4-FITC antibodies. The following controls should be considered essential:

For mouse-derived RFC4 antibodies used on mouse tissues, Mouse-on-Mouse blocking reagents should be employed to reduce background in IHC and ICC experiments . This is particularly important when using monoclonal mouse antibodies like the OTI1A8 clone on mouse samples.

When analyzing cell cycle-dependent expression of RFC4, additional controls should include cell cycle markers and DNA content analysis to correlate RFC4 levels with specific cycle phases.

How can researchers address high background when using RFC4-FITC antibodies?

High background is a common issue when working with RFC4-FITC antibodies, particularly in immunofluorescence applications. Several strategic approaches can help minimize this problem:

• Optimize blocking conditions:

  • Extend blocking time to 1-2 hours at room temperature

  • Test different blocking agents (5% BSA, 5-10% normal serum, commercial blocking buffers)

  • For mouse-derived antibodies on mouse tissues, use specialized Mouse-on-Mouse blocking reagents as recommended for OTI1A8 clone

• Adjust antibody concentration:

  • Perform titration series to identify minimal effective concentration

  • Begin with higher dilutions (1:200-1:500) and adjust based on signal-to-noise ratio

  • Consider using purified antibody preparations (>95% purity through Protein G purification)

• Improve washing protocol:

  • Increase number of washes (minimum 3-5 washes)

  • Extend wash duration (10-15 minutes per wash)

  • Add 0.05-0.1% Tween-20 to wash buffers to reduce non-specific binding

• Adjust fixation and permeabilization:

  • Over-fixation can increase autofluorescence; optimize fixation times

  • Excessive permeabilization may increase non-specific binding

  • Consider reduced-formaldehyde protocols for sensitive epitopes

• Address autofluorescence:

  • Use Sudan Black B (0.1-0.3%) to quench autofluorescence in tissues

  • Consider spectral unmixing on confocal systems

  • Use commercially available autofluorescence quenching reagents

If working with FITC antibodies in tissues with high intrinsic autofluorescence, consider alternative conjugates with longer emission wavelengths like Janelia Fluor 669 which is available for the same RFC4 antibody clone .

What are the common causes of signal variability with RFC4-FITC antibodies and how can they be minimized?

Signal variability can significantly impact the reproducibility and reliability of experiments using RFC4-FITC antibodies. Understanding and controlling sources of variability is essential for generating consistent, high-quality data.

Common causes of signal variability include:

To minimize variability:

• Implement standardized protocols with precise timing for all steps

• Process all samples for comparative studies simultaneously

• Include internal standards or reference samples across experiments

• Use automated systems for staining where possible

• Maintain consistent imaging parameters (exposure time, gain, laser power)

• Consider alternative formulations with greater stability (such as those containing 50% glycerol)

• Use proper buffer systems (0.01M PBS, pH 7.4)

How can RFC4-FITC antibodies be used to study DNA replication dynamics in cancer cells?

RFC4-FITC antibodies offer powerful tools for investigating DNA replication dynamics in cancer cells, where replication stress and aberrant replication are hallmarks of malignancy. Advanced research applications include:

• Co-localization studies with replication machinery components:

  • Combine RFC4-FITC with antibodies against PCNA, RFC2-5, or DNA polymerases

  • Use super-resolution microscopy to visualize replication factories

  • Quantify co-localization coefficients at different cell cycle stages

• Cell cycle-dependent RFC4 expression analysis:

  • Synchronize cells at different cell cycle phases (G1, S, G2/M)

  • Measure RFC4-FITC fluorescence intensity by flow cytometry or microscopy

  • Correlate with EdU or BrdU incorporation to identify actively replicating cells

• Response to replication stress:

  • Treat cells with replication stress inducers (hydroxyurea, aphidicolin)

  • Analyze changes in RFC4 localization, abundance, or post-translational modifications

  • Combine with γH2AX staining to correlate with DNA damage sites

• Cancer cell line panels:

  • Compare RFC4 expression across cancer cell lines with different proliferation rates

  • Correlate with tumorigenic potential or therapeutic resistance

  • Use as a potential biomarker for replication stress susceptibility

• Therapeutic intervention studies:

  • Monitor RFC4 dynamics before and after treatment with targeted therapies

  • Assess changes in replication complex formation following drug exposure

  • Identify potential mechanisms of resistance to replication-targeting drugs

For quantitative analysis of RFC4 in diverse cancer cell types, the monoclonal RFC4 antibody clone OTI1A8 with FITC conjugation has been validated across multiple human cancer cell lines including HeLa, 293T, and K562 .

What multiplex strategies work effectively with RFC4-FITC antibodies?

Effective multiplex imaging strategies with RFC4-FITC antibodies enable simultaneous visualization of RFC4 alongside other proteins of interest, providing insights into complex molecular interactions and cellular contexts. When designing multiplex experiments:

• Compatible fluorophore selection:

  • Pair FITC (excitation: 495 nm, emission: 519 nm) with spectrally distinct fluorophores

  • Recommended combinations include:

    • FITC + Cy3/TRITC + Cy5/AlexaFluor647

    • FITC + PE/Texas Red + APC

  • Avoid fluorophores with significant spectral overlap (GFP, BODIPY-FL)

• Sequential staining protocols:

  • For multiple primary antibodies from the same host species:

    • Apply tyramide signal amplification (TSA) between sequential antibody applications

    • Use direct conjugates where possible to avoid secondary antibody cross-reactivity

  • Consider Zenon labeling technology for simultaneous use of multiple mouse antibodies

• Panel design considerations:

  • Combine RFC4-FITC with markers of:

    • DNA damage response (γH2AX, 53BP1)

    • Cell cycle phases (Cyclin proteins, Ki-67)

    • Replication complex components (PCNA, RFC2-5)

  • Place brightest fluorophores on least abundant targets

• Alternative conjugates:

  • When FITC channel is unavailable or suboptimal, consider alternative conjugates:

    • RFC4 antibody with Janelia Fluor 669 for far-red detection

    • Use secondary antibody approach with spectrally distinct conjugates

For researchers requiring multiplexing in tissues with high autofluorescence in the FITC channel, the same RFC4 antibody clone (OTI1A8) is available with far-red Janelia Fluor 669 conjugation, which can provide superior signal-to-noise ratios in challenging samples .

How do RFC4-FITC antibodies perform in live cell imaging applications?

Live cell imaging with RFC4-FITC antibodies presents unique challenges and opportunities for studying dynamic DNA replication processes. While most applications utilize fixed cells, live cell approaches can be implemented with specific considerations:

• Antibody delivery methods:

  • Microinjection of RFC4-FITC antibodies (1-2 mg/ml)

  • Cell-penetrating peptide conjugation

  • Electroporation or nucleofection protocols

  • Temporary membrane permeabilization with streptolysin O

• Optimization considerations:

  • Antibody concentration: Lower concentrations (1-5 μg/ml) minimize interference

  • Imaging buffer composition: Supplement with antioxidants to reduce phototoxicity

  • Exposure settings: Use minimal excitation intensity and duration

  • Time-lapse intervals: Balance temporal resolution against photobleaching and phototoxicity

• Alternative approaches:

  • CRISPR/Cas9 knock-in of fluorescent tags to endogenous RFC4

  • Transient expression of RFC4-GFP fusion proteins

  • Correlative live/fixed imaging: live cell imaging followed by fixation and RFC4-FITC staining

• Technical limitations:

  • FITC photobleaching occurs rapidly in live cell conditions

  • Antibody binding may interfere with protein function

  • Intracellular antibody concentration is difficult to control

  • Nuclear localization requires specialized delivery strategies

• Controls and validation:

  • Confirm cell viability throughout imaging (membrane-impermeant dyes)

  • Validate antibody specificity in fixed cells before attempting live cell approaches

  • Compare with alternative visualization methods (e.g., fluorescent protein fusions)

For researchers specifically interested in live cell dynamics of RFC4, alternative strategies such as expressing fluorescent protein-tagged RFC4 may provide more reliable results than direct antibody approaches.