Detection reagent and detection method of fxa
A technology for detecting reagents and metal-binding proteins, applied in the field of biochemical detection, can solve problems such as easy generation of errors, and achieve the effects of simple operation, good repeatability and strong binding force
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Embodiment 1
[0031] Embodiment 1 detection reagent preparation
[0032] Metal binding proteins AHP, Tb(NO 3 ) 3 ·5H 2 O and dissolved in Tris-HCl buffer solution at pH 7.4. Use deionized water to adjust the concentration of metal binding protein AHP to 5nmol / L, rare earth ion Tb 3+ Concentration is 10nmol / L, Tris-HCl concentration is 200nmol / L, mix well for 5 minutes, add magnesium sulfate solution, adjust Mg 2+ The concentration was 10 μM and the reaction was mixed thoroughly for 5 minutes
Embodiment 2
[0033] The detection of embodiment 2 detection reagent
[0034] Fluorescent detection of reagents in different states ( figure 1 ) and electrophoretic detection ( Figure 2~3 ).
[0035] Tb 3+ The fluorescence itself is very weak, Tb 3+ After adding AHP, the fluorescence is greatly enhanced, and the addition of Mg 2+has no effect on it. FXa cannot make Tb 3+ Fluorescence enhancement, FXa added to AHP-Tb 3+ -Mg 2+ Tb 3+ Fluorescence decreased, but still well above Tb 3+ -Mg 2+ Medium fluorescence intensity. Add low concentration BSA to AHP-Tb 3+ -Mg 2+ Medium Tb 3+ The fluorescence remained basically unchanged, indicating that the low concentration of impurity proteins had no effect on the system.
[0036] figure 2 Electrophoresis environment is 1mMMg 2+ , 1 channel is AHP, 2 channels are AHP+FXa, 3 channels are AHP+Tb 3+ FXa, lane 4 is FXa. Among them, in lanes 2 and 3, AHP and FXa were completely combined to form a band. image 3 No Mg in the electrophore...
Embodiment 3
[0038] Example 3 Sample Detection
[0039] 1. Determine the appropriate sample dilution ratio. Due to the high concentration of impurity proteins will affect the AHP and Tb at 280nm 3+ Fluorescence resonance energy transfer between, so a suitable dilution ratio is needed to meet the experimental requirements.
[0040] Mix according to the detection reagent and the FXa-depleted plasma volume ratio of 50:1, 150:1, 200:1 (number 1, 2, 3 respectively). At the same time, the fluorescence of the detection reagent (No. 4) was used as a control to calculate the error. After 20 minutes, inject 200 microliters of samples into a black 96-well plate, set the time-resolved fluorescence endpoint method mode, and automatically adjust the gain multiplier of the microplate reader. Delay: 50μsec, acquisition time: 1950μsec, excitation wavelength 280nm, detection wavelength 546nm The fluorescence intensity( Figure 4 ).
[0041] The results are shown in Table 1:
[0042] Table 1 Fluorescen...
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