Method for detecting interaction of proteins
A protein interaction and detection method technology, applied in biological testing, material inspection products, fluorescence/phosphorescence, etc., can solve the problems of long experimental period, low sensitivity, cumbersome operation, etc., and achieve high-resolution detection and research effects
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Embodiment 1
[0029] The construction of embodiment 1 bacterial two-hybrid system
[0030] Using the Escherichia coli K12 genome as a template, primers for pal and tolB genes were designed and synthesized in Yushengong, and pal and tolB genes were obtained by PCR. They were respectively inserted into the EcorI and XhoI restriction sites of bacterial two-hybrid vectors pUT18 and pKT25 to obtain pUT18-pal and pKT25-tolB. The two vectors carrying pal and tolB were co-transformed into the reporter bacterium E.coli BTH101 to complete the construction of the bacterial two-hybrid system. The successful establishment of the Pal-TolB bacterial two-hybrid system was confirmed by routine blue-white screening and colorimetric quantitative methods, and was verified by traditional biological experiments such as Western Blot, co-immunoprecipitation, and fluorescence microscopy.
Embodiment 2
[0031] The cultivation of embodiment 2 two-hybrid bacteria and the expression of interacting protein
[0032] Pick a single colony of E.coli BTH101+pUT18-pal+pKT25-tolB in 2mL LB medium, add the inducer IPTG to a final concentration of 0.5mM, and culture at 30°C and 250rpm for 18h to induce the expression of Pal and TolB. The interaction of the two proteins promotes the transcription and expression of the reporter gene product β-gal. At the same time, BTH101pKT25+pUT18; BTH101pKT25-TolB+pUT18; BTH101pKT25-+pUT18-Pal were set as negative controls.
Embodiment 3
[0033] Example 3 Determination of β-gal enzyme activity by UV-visible spectrophotometry to determine protein interaction
[0034] The OD600 values of the four bacterial solutions in Example 2 were measured with a spectrophotometer, and the OD600 of the four bacterial solutions were basically consistent by adjusting the amount of M63 medium added. Using the Miller method, ONPG was used as a substrate, and the β-gal enzyme activity of the four bacteria was measured on a UV-visible spectrophotometer (set DU800, kinetic / time, analytical 420nm, Scan time 20min, interval 2min), so as to determine the protein interaction situation.
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