During large-scale manufacturing of biologics, nucleases are widely employed for targeted removal of host cell residual DNA. The activity level of these nucleases directly impacts product safety and quality control. Therefore, reliable detection methods are essential for: quality verification of nuclease raw materials, precise calibration of dosing, stability monitoring during storage and usage, and detection of residual nuclease impurities in final products. This testing step has become a critical component of the quality control system for biologics.

This kit utilizes a specially designed, highly sensitive fluorescent substrate to provide a stable, direct, and user-friendly solution for nuclease activity quality control, capable of meeting diverse testing requirements.

The kit enables highly sensitive detection of multiple non-specific nucleases. It includes a separately packaged, high-concentration NaCl reagent, allowing flexible adjustment of salt concentration in the reaction system according to the optimal conditions of different nucleases, thereby ensuring assay specificity and accuracy.

The kit employs a simplified, standardized workflow: test samples are serially diluted, then co-incubated with the fluorescent substrate at a constant temperature for 5–60 minutes. After adding stop solution to quench the reaction, fluorescence intensity is measured using a fluorescence detection instrument. Nuclease activity is quantified based on a linear regression curve generated from a series of standard references. Results are expressed as the amount of fluorescent product generated per unit time, providing a direct readout of enzyme activity.

This kit uses an endpoint detection method, eliminating the need for complex kinetic analyses such as 4-parameter logistic (4-PL) curve fitting, significantly improving testing efficiency and enabling high-throughput screening applications. Following reaction termination, the fluorescent signal remains stable for at least 10 minutes and is compatible with mainstream fluorescence detection platforms—including fluorescence microplate readers, real-time fluorescent PCR instruments, and fluorescent micro-spectrophotometers—accommodating various laboratory equipment configurations.

Specifications

Components

Storage

All components are shipped on dry ice.This product should be stored at -25~-15℃ protected from light for 1 year.

Self-provided materials

1. Nuclease-free pipettes, tips, microcentrifuge tubes, and black opaque-bottom 96-well microplates.

2. A multifunctional fluorescence microplate reader, real-time fluorescent PCR instrument, or fluorescent micro-spectrophotometer.

Instructions

This kit supports three data acquisition approaches. Please select the method appropriate for your experimental needs:

1. Direct Observation Method

Suitable for rapid qualitative assessment (e.g., nuclease contamination screening). This method is intuitive and simple: nuclease-contaminated samples emit characteristic fluorescence under UV light, with intensity proportional to nuclease concentration. However, this approach lacks quantitative precision and should not be used for quantitative evaluation.

2. Endpoint Fluorescence Method

For quantitative results, endpoint fluorescence analysis directly measures accumulated fluorescence intensity at reaction termination or determines enzyme activity units under defined conditions, enabling precise quantification of nuclease activity. The optimized design of this kit ensures measurement reliability and reproducibility.

3. Real-Time Fluorescence Kinetic Method

Using a real-time fluorescence microplate reader, continuous monitoring of the reaction allows accurate determination of enzymatic reaction rates and comparative activity analysis. When performing activity-based nuclease quantification with this method, a calibrated and stability-confirmed nuclease reference standard must be used. Such standards can be directly calibrated using the endpoint fluorescence method.

Experimental Protocol

1. Pre-Experiment Preparation

(1) Clean all equipment and consumables to avoid nuclease and protease contamination.

(2) For fluorescence grating-based microplate readers or other fluorescence detectors, set excitation/emission wavelengths to Ex/Em: 488/525 nm.

(3) For filter-based fluorescence detection systems, ensure optical filters are compatible with FAM detection (typically Ex/Em: 485–495 nm / 515–525 nm).

(4) Before use, thaw all components and equilibrate at room temperature for 5 minutes. Vortex thoroughly before use.

2. Procedure (Endpoint Fluorescence Method)

2.1 Dilution Buffer (DB) Preparation

Dilute the 10X Buffer (Low Salt) to 1X using nuclease-free water to prepare Dilution Buffer (DB). The provided 4.5 M NaCl solution may be used to adjust the salt concentration in DB (initial salt concentration: 50 mM; adjustable up to 500 mM).

2.2 Substrate Working Solution (SW) Preparation

Dilute the Nuclease Substrate 50-fold in DB to prepare Substrate Working Solution (SW).

2.3 Standard Curve Dilution

Perform serial dilutions of the reference standard in DB according to the table below:

2.4 Sample Preparation

Dilute test samples in DB to the desired concentration. For initial testing of unknown samples, perform multiple 10-fold serial dilutions and test each dilution.

2.5 Sample Loading

Three sample types are tested: standards, negative controls, and test samples. All sample types must be run in triplicate for statistical reliability.

(1) Standards: Add 100 μL of each STD1–STD9 per well.

(2) Negative Control: Add 95 μL SW + 5 μL DB per well.

(3) Test Samples: Add 95 μL SW + 5 μL sample per well.

2.6 Mixing and Incubation

Thoroughly mix the 96-well plate and incubate at 37°C for 5–60 minutes. Accurately record the incubation time for enzyme activity calculation.

2.7 Reaction Termination

Add 50 μL Stop Solution to each well to terminate the reaction, then immediately proceed to detection.

2.8 Fluorescence Measurement and Data Analysis

2.8.1 Fluorescence Acquisition

Use a calibrated fluorescence detector to acquire data. Valid data must meet the following criteria:

(1) Negative Control: Fluorescence value ≤ that of STD9. Higher values suggest nuclease contamination.

(2) Valid Fluorescence Range: For quantitative assays, acceptable sample fluorescence must fall between STD1 and STD9. If a sample’s fluorescence exceeds STD1, retest after appropriate dilution. For qualitative assays, the limit of detection is defined as twice the solvent control value.

2.8.2 Standard Curve Construction

Plot FAM content (y-axis) against fluorescence intensity (x-axis) for the reference standards and perform linear regression to obtain the equation y = ax + b. The correlation coefficient (R²) must be ≥ 0.995.

2.8.3 Nuclease Activity Calculation

Substitute the sample’s fluorescence value into the standard curve to calculate the amount of product generated (F, in pmol) during the reaction by the diluted sample volume added. Divide F by the sample volume (V, in μL) and reaction time (T, in minutes) to obtain the volumetric enzyme activity of the diluted sample. Multiply by the dilution factor (D) to determine the volumetric nuclease activity of the original sample.

Use the following formula to calculate the original sample’s enzyme activity:

A: Volumetric enzyme activity (pmol/min/μL)

V: Sample volume added (standard protocol: 5 μL)

F: Product amount calculated from the standard curve (pmol)

T: Reaction time (minutes)

D: Sample dilution factor

2.8.4 Enzyme Unit Quantification

Enzyme activity units may be determined using either of the following validated approaches:

(1) Direct Calculation Method: If the reaction conditions defined in this assay match the official definition of one enzyme unit, and the assay method aligns with the unit definition, the calculated activity value may be directly reported as enzyme units.

(2) Comparative Calibration Method: First, assay a reference standard with known enzyme units under identical conditions to establish a standard curve or conversion factor between measured activity and defined units. Then, assay the test sample under the exact same conditions and use the established curve or factor to convert its measured activity into enzyme units.

Notes

1. Read this manual carefully before use. Perform experiments according to standardized protocols for sample handling, reaction mixture preparation, and pipetting. Pipette quickly and gently into the bottom of microplate wells to avoid splashing.

2. Use this product only within its expiration date. Do not mix reagents from different lots.

3. Protect Components A and B from prolonged light exposure to prevent photobleaching.

4. Wear a lab coat and disposable gloves for safety.

5. This product is intended for research use only.

Documents:

Safety Data Sheet

41315_MSDS_HB251205_EN.PDF

Manuals

41315_Manual_Ver.EN20251204.pdf