UniOne® TR-FRET His Check kit

The kit employs homogeneous time-resolved fluorescence technology (TR-FRET) to quantify His-tagged proteins. This kit can be used both to confirm whether the His-tag motif of a protein is recognized by anti-His antibodies and to estimate the concentration of His-tagged proteins.

As shown in the figure, the protein with aHis tag is labeled with the Acceptor, namely Ac-Protein His Tag, while the antibody that recognizes the His tag is labeled with Eu, i.e., Eu-anti-His. After the antibody binds to the His tag on the protein, the distance between the donor and acceptor decreases, and excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) to the acceptor antibody, causing the acceptor antibody to emit a signal at a wavelength of 665 nm. When other His-tagged proteins are present, they compete with Ac-Protein His Tag for binding to Eu-anti-His, preventing FRET signals from occurring; the higher the concentration of these other His-tagged proteins, the greater the competition with Ac-Protein His Tag.CombineThe stronger the Eu-anti-His binding capacity, the lower the signal. The signal is directly proportional to the binding of Ac-Protein His Tag and Eu-anti-His, and no washing steps are required.

1. Reagent Preparation

1.1 Before use, thaw all reagents at room temperature (allow them to equilibrate at room temperature for at least 30 minutes). The reaction system in a 384-well plate is 20 μL (the amount of reagents for the reaction system is shown in the table below). Calculate the required volume for your experiment before preparation and prepare accordingly; the following preparation is for reference only, using 500 reactions as an example.

Table 1. Reagent Preparation

1.2 Gradient Dilution of Samples to Be Measured

Taking His Tag Control as an example, the diluent is 1× Detection buffer. To reduce the impact of matrix effects, it is recommended to dilute with a solution that has the same matrix as the sample to be tested; moreover, the concentration of the sample should be adjusted according to its actual level.

Table 2. Gradient Dilution of Positive Control His Tag (Adjust According to Actual Conditions)

2. Sample Addition and Controls

2.1 Sample Wells: Add 10 μL of the sample to be tested (gradient dilution), 5 μL of Eu-anti-His working solution, and 5 μL of Ac-Protein His Tag sequentially into the 384-well plate.

2.2 Positive Control: Add 10 μL of 1× Detection buffer, 5 μL of Eu-anti-His working solution, and 5 μL of Ac-Protein His Tag sequentially into the 384-well plate.

2.3 Negative control: Add 15 μL of Detection buffer and 5 μL of Eu-anti-His working solution sequentially into the 384-well plate.

After all samples have been added, centrifuge, seal with a plate cover, and incubate at room temperature for 2 hours.

3.Detection

Detect on an ELISA reader compatible with TR-FRET. Use an excitation wavelength of 320/340 nm, and measure emission wavelengths at 620 nm and 665 nm.

[Result Calculation]

1) Calculate the signal value (Ratio): Multiply the 665nm fluorescence signal by the 620nm fluorescence signal, then multiply by 10000.

Ratio = (665/620) ×10000

2) Calculate the Net signal based on the signal value:

Net signal = (Std-NC)/NC×100

3) Calculate CV (%):

CV (%) = Standard Deviation/Mean Ratio × 100%

[Data Example]

The following data cannot replace the data obtained in experiments; it is only for illustration, and results may vary depending on the plate reader. 

Note: Microplates are recommended (384-well plates, white, shallow wells)