Biothiol detecting composition comprising redox regulation protein
A technology for regulating proteins and biological thiols, which can be used in biological testing, biomaterial analysis, peptides containing affinity tags, etc., and can solve problems such as limiting repeatability and accuracy
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Examples
Embodiment 1
[0097]Example 1: Confirmation of reactivity between OhrR protein and Cys, Hyc or GSH
[0098] Real-time DNA binding activity of OhrR was measured using fluorescence anisotropy (FA).
[0099]
[0100] Buffer: 20mM Tris (pH 8.0) 150mM NaCl, 5% Glycerol (v / v)
[0101] DNA concentration: 50nM
[0102] OhrR concentration: 300nM
[0103] CHP concentration: 3 μM
[0104] Measuring time: measure once every 10s
[0105] Measurement conditions: ex 492nm; slit width 15nm, em 520nm; slit width 20nm, integration time 1s
[0106] DNA sequence:
[0107] Template DNA: 5'-TAC AAT TTA ATT GTA TAC AAT TTA ATT GTA-3' (SEQ ID NO: 1)
[0108] Complementary DNA: 5'-TAC AAT TTA ATT GTA TAC AAT TTA ATT GTA-3' (SEQ ID NO: 2)
[0109] OhrR sequence: cloned directly into a Bacillus subtilis strain.
[0110]
[0111]
[0112] After dissolving in 3 mL of buffer under the experimental conditions, the binding activity between OhrR and fluorescent (6FAM, 6-carboxyfluorescein)-labeled OhrR-bound...
Embodiment 2
[0115] Example 2: Confirmation of binding between OhrR protein and target DNA using electrophoresis
[0116] Fluorescent probe (FAM)-bound double-stranded (ds) DNA (200nM) [SEQ ID NO: 1 and 2] was mixed with image 3 The concentration of OhrR indicated in was mixed and reacted at room temperature for 30 minutes, and then the dsDNA band was detected by using a polyacrylamide gel (7%) electrophoresis (25mA, 30min) with a fluorescence spectrometer (KIF-300, Korea Lab Tech, Korea) . It can be seen that the fluorescence band of dsDNA shifts upward from OhrR concentration of about 1.6 μM (about 8 times the concentration of DNA).
[0117] Unlike the FA measurement method described in Example 1, PAGE electrophoresis does not yield real-time measurements of protein-DNA binding and requires a large amount of protein to bind OhrR and DNA (thus, the actual binding constant cannot be measured by this method), but this This method can be used as a method to easily identify binding without...
Embodiment 3
[0118] Example 3: Experiments using photorefractive index to measure the process of binding between dissociated DNA and OhrR protein without a fluorescent label
[0119] The degree of binding between DNA and protein on the surface of the photosensor (optical fiber) was measured using an instrument for biolayer interferometry (Blitz, Fortebio, USA).
[0120]
[0121] Binding buffer, washing buffer: TBS (Tris 20mM, NaCl 150mM)
[0122] DNA binding time, OhrR binding time: 2 minutes
[0123] Washing time: 30 seconds
[0124] DNA concentration: 2.5 μM
[0125] OhrR concentration: 50 μM
[0126] CHP concentration: 100 μM
[0127]
[0128] First, biotin-conjugated dsDNA [SEQ ID NO: 1 and 2] was dissolved in TBS buffer at the above concentrations, and the biotin-conjugated dsDNA and streptavidin-coated photosensors were allowed to flow by letting the resulting mixture flow for 120 seconds (fiber optics) bound, washed with buffer, and OhrR protein was then associated (associa...
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