Protein detection kit and detection method
A protein detection and detection method technology, applied in the field of trace protein molecular detection, can solve the problems of false positive signals caused by non-specific combination of nucleotide sequences, affecting Taq polymerase activity and detection efficiency, affecting reaction efficiency, etc., to avoid The effect of reduced sensitivity, good specificity, and simple preparation method
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Embodiment 1
[0045] Embodiment 1: Synthetic reaction of antibody-oligonucleotide probe
[0046] The synthesis steps of the antibody-oligonucleotide probe of the protein detection kit include the following:
[0047] Step 1. Resuspend the matching monoclonal antibody (mAb) or polyclonal antibody (pAb) divided into two parts in phosphate buffer saline (PBS) at 1 mg / mL. The 100K ultrafiltration tube was equilibrated four times with conjugation buffer, and the equilibration conditions were: 400 μL / well, 3000 rpm, 1 minute×4. The composition of the conjugation buffer is: 100 mM sodium phosphate, 150 mM sodium chloride, 1-5 mM ethylenediaminetetraacetic acid (EDTA), pH=7.2. The role of EDTA is to help chelate divalent metals, thereby reducing the formation of disulfide compounds in sulfur-containing proteins.
[0048] Step 2, the sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate sodium (sulfo-SMCC sodium) with a molecular weight of 436.37 in ultrapure water (ddH 2 After dissol...
Embodiment 2
[0060] Example 2: Preparation of qPCR tubes pre-coated with antibodies treated with glutaraldehyde
[0061] The preparation method of the qPCR tube of the pre-coated antibody treated with glutaraldehyde of the protein detection kit comprises the following steps:
[0062] Step 1. Dilute the glutaraldehyde solution (glutaraldehyde is a bifunctional reagent through its aldehyde group to the amino group on the immunoglobulin covalently) to 0.8% with deionized water, and add it to the bottom of the eight-tube tube, Add 25 μL to each tube and place in a 37°C water bath for 5 hours. Then wash three times with deionized water.
[0063] Step 2. Add 25 μL of antibody with a concentration of 1 μg / ml (polyclonal antibody is better than monoclonal antibody) coating buffer, and incubate overnight at 4°C or 37°C for 1 hour. The composition of the coating buffer is: 15mM Na 2 CO3 , 35mM NaHCO 3 , pH=9.6.
[0064] Step 3. After the binding is completed, wash with washing buffer three time...
Embodiment 3
[0065] Embodiment 3: solid adjacent position extension reaction
[0066] Figure 4 It is a schematic diagram of the solid adjacent extension reaction of the present application.
[0067] It can be seen from the figure that the solid adjacent extension reaction includes:
[0068] S301. Antibody pre-coated PCR tube. Specifically as described in Example 2, the PCR tube 31 is pre-coated with the antibody 32 . Then, add the target protein 33 to be tested.
[0069] S302. Capturing the target protein to be detected. On the solid phase interface 34 treated with glutaraldehyde, the antibody 32 captures the target protein 33 to be detected.
[0070] S303, adding antibody-oligonucleotide probes. A pair of antibody-oligonucleotide probes 35 is formed by covalently combining two monoclonal antibodies (or polyclonal antibodies) with different epitopes and oligonucleotides that can form complementary chains. Specifically as described in Example 1.
[0071] S304. The probe recognizes ...
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