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dPEG biotinylation plays a vital role in our production and life. What are the purification and related procedures of dPEG biotinylation? Next, let us take a look.
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l Expression and purification of 3-PBA Nanobody
l 3-PBA Nanobody dPEG biotinylation
l ELISA program based on dPEG biotinylation nano antibody
Expression and purification of 3-PBA Nanobody
Pick a single colony of TOP 10F' Escherichia coli carrying pComb3x-3-PBA nano-dPEG biotinylation antibody expression vector, and inoculate it into 10 mL of LB medium containing ampicillin (100 ug/mL) and tetracycline hydrochloride (100 ug/mL) (LB-Amp-TC) medium shake (the shaking conditions below are all 37 ℃, 250 r/min) overnight. The overnight bacteria were expanded to 1L of LB-Amp-TC, and when the OD value reached 0.6-0.8, IPTG solution (1 mmol) was added to induce expression for 12 h. Centrifuge the bacterial solution, discard the supernatant, add 5 mL TES solutions (12.1g'Tris, 0.093 g EDTA, 85.575 g sucrose, 500 mL water, adjusted to pH 8.0 with hydrochloric acid), stir until the precipitate is completely dispersed, and freeze at -80°C, Thaw at 37°C, repeat freeze-thaw 3 times. After the last thawing, add 12 mL of pure water and 3 mL of LTES solution, put it in a shaker at 10°C, shake at 180 r/min for 30 minutes, and centrifuge to collect the supernatant. For dPEG biotinylation, 10 and 20 mmol imidazole-PBS were used to wash away unbound non-specific components, and then 50, 100, and 200 mmol imidazole-PBS were used to elute the nano-dPEG biotinylation antibody and collect the eluate. Take an appropriate amount of flow-through solution and imidazole eluate of different concentrations for SDS-PAGE detection. Dialysis Nanobody eluate with 0.01 mol/L PBS to remove imidazole, ultrafiltration tube to concentrate the protein solution to above 1 mg/mL, and store at -80 °C.
3-PBA Nanobody dPEG biotinylation
Weigh 5 mg of biotin—NHS dissolved in 200 uL DMSO, add 5 mg of nano-dPEG biotinylation antibody dissolved in pH 8.0 PBS buffer, react overnight at 4°C, and dialyze the unreacted biotin with PBS the next day— NHS. After the dialysis, ELISA was performed to verify the activity of the antibody after biotinylating. Place the rest in a -80 ℃ refrigerator for later use.
ELISA program based on dPEG biotinylation nano antibody
Dilute the original dPEG biotinylation coating with carbonic acid buffer to the desired concentration, add 100 uL/well to the microtiter plate, and let it stand overnight in a 37°C water bath. Wash twice the next day, each time with 300 uL of PBS containing Tween-20 with a volume fraction of 0.005%, and then pat dry the plate on absorbent paper. Add 120 uL of Porin Sealing Solution, let it stand at 37°C for 3 hours, then pour out the liquid in the well, pat dry on absorbent paper, dry at 37°C before use, or keep it sealed at 4°C for later use. Dilute the 3-PBA nano-dPEG biotinylation antibody and 3-PBA standard drug with PBS to the required concentration, add 50 uL antibody diluent and 50 puL.3-PBA standard solution to each well of the microplate, and incubate at 37°C for 40 min. Wash the plate 5 times and pat dry. Dilute dPEG biotinylation to a suitable concentration with PBST, add 100 uL to each well, incubate at 37°C for 30 min, wash the plate 5 times, and pat dry. After mixing TMB solution A and B in equal volumes, add 100 uL to each well, react at 37°C for 10 min, then add 50 puL stop solution to each well, and measure the absorbance of each well of dPEG biotinylation at 450 nm with a microplate reader value.
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