Protein for being combined with TAFIa/ai and application thereof, and kit for detection of TAFIa/ai content
A kit and protein technology, applied in the biological field, can solve the problems of complicated operation of detection methods, unstable detection results, low detection sensitivity, etc., and achieve the effects of improving detection sensitivity, low detection limit and low cost.
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Embodiment 1
[0058] The preparation of embodiment 1 TAFIa / ai chemiluminescent quantitative detection reagent
[0059] (1) Construction of PGT1 protein
[0060] Entrust a technical service company to synthesize the PGT1 protein sequence designed in the present invention according to the codon preference of Escherichia coli BL21. The protein sequence is as follows (SEQ ID NO.1):
[0061] EQHADPICNKPCKTHDDCSGAWFCQACWNSARTCGPYVGMKLYFSRNPNPRLAVAIARYLETKLDFEFASPFAAGQMEKFRRLNPNLSLPILVDDEGKSLWEADAIACRLSRHAHSDFWRTGDDEPEMIRWLSWGKEHFALACDTVHFERGTKQRYGIGPIDQKRVEEGLNQFHTAAAMLDAVLAERQWLVGNSVSYADFRMATFLPFNDAARLPLDDYPSVSRWYRRLEDIDAWRDPFKGMDAPELPPVPQMAVADQREQYDPVCHKPCSTQDDCSGGTFCQACWRFAGTCGPYVHHHHHH。
[0062] The physical map of the obtained vector is as figure 1 shown.
[0063] The prepared protein was subjected to electrophoresis, and the results were as follows: figure 2 Shown: each lane is ①purified PGT1 protein; ②purified PGT1 protein; ③purified PGT1 protein; ④unpurified PGT1 protein; M protein ma...
Embodiment 2
[0090] Example 2 TAFIa / ai Chemiluminescent Quantitative Detection Method I
[0091] (1) The model of the chemiluminescence instrument is BPCL weak luminescence measuring instrument. The detection cell temperature was set at 37°C.
[0092] (2) Add 150 μL of the reagent I-a provided in step (6) of Example 1 of the present invention, 150 μL of reagent I-b provided in step (9) of Example 1 of the present invention and 5 μL of the standard in the detection tube, mix After homogenization, place in the detection pool and incubate for 10 min.
[0093] (3) Magnetic separation, remove the supernatant. Inject 50 μL each of liquid A and liquid B in the chemiluminescent substrate I provided in step (11) of Example 1 of the present invention into the detection tube in sequence, and start recording the luminescence intensity for 300 s. In this experiment, the photon count corresponding to the peak area of the chemiluminescence kinetic curve was collected.
[0094] (4) Establish a stand...
Embodiment 3
[0098] Example 3 TAFIa / ai Chemiluminescent Quantitative Detection Method II
[0099] (1) The model of the chemiluminescence instrument is BPCL weak luminescence measuring instrument. The detection cell temperature was set at 37°C.
[0100] (2) Add 150 μL of the reagent II-a provided in step (7) of Example 1 of the present invention, 150 μL of reagent II-b provided in step (10) of Example 1 of the present invention and 5 μL of the standard in the detection tube, mix After homogenization, place in the detection pool and incubate for 10 min.
[0101] (3) Magnetic separation, removing the supernatant, and adding 300 μL of the chemiluminescent cleaning solution provided in step (13) of Example 1 of the present invention. Remove the magnetic field, mix well and incubate in the detection cell for 5 minutes.
[0102] (4) Inject the chemiluminescent substrate II provided in step (12) of Example 1 of the present invention into the detection tube, start recording the luminescence inte...
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