A method for rapid fluorescent labeling of proteins
A fluorescent labeling, protein technology, applied in the field of bioengineering, to achieve the effect of fast binding, convenient use and high specificity
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Embodiment 1
[0016] Application of this labeling method on soluble proteins in solution
[0017] a. The green fluorescent protein expressed in Escherichia coli is shown as sequence 1 in the sequence table; expression strain: BL21*(DE3), carrier: pET-15b (Escherichia coli protein expression vector). Shake at 37°C until OD=0.8, add 1 mM IPTG (Isopropyl β-D-Thiogalactoside), and induce at 37°C for 4 hours. Collect bacteria by high-speed centrifugation;
[0018] b. Resuspending the cells with Ni A buffer solution and then autoclaving the bacteria, wherein the Ni A buffer solution includes 20 mM Tris (abbreviated as Tris) and 200 mM NaCl, and the pH is 8.0. The supernatant was collected by high-speed centrifugation, passed through a Ni column, and eluted with a gradient from 60 mL to 200 mM imidazole. The collected fractions were concentrated with an ultrafiltration tube and exchanged from Ni A buffer to QA buffer, wherein the QA buffer included 20 mM tris with a pH of 8.0. Pass through the ...
Embodiment 2
[0024] Application of this labeling method on cell membrane surface proteins
[0025] a. The green fluorescent protein expressed in Escherichia coli is shown as sequence 1 in the sequence table; expression strain: BL21*(DE3), carrier: pET-15b. Shake at 37°C until OD=0.8, add 0.5mM IPTG, and induce at 19°C for 20h. Collect bacteria by high-speed centrifugation;
[0026] b. Resuspend the bacteria with Ni A buffer solution and then autoclave the bacteria, wherein the Ni A buffer solution includes 20 mM Tris (abbreviated as Tris) and 200 mM NaCl, and the pH is 8.0. The supernatant was collected by high-speed centrifugation, passed through a Ni column, washed with 20mM imidazole, i.e. 10% Ni B buffer, and then eluted with 200mM imidazole, i.e. 40% Ni B buffer, wherein the Ni B buffer included 20mM Tris (abbreviated as Tris), 200mM NaCl, 500mM imidazole, pH 8.0. The collected fractions were concentrated to about 6 mL, desalted to Q A buffer, where Q A buffer was 20 mM tris, and i...
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