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Purification and related procedures of dPEG biotinylation

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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.

Here is the content:

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.

The above is about the purification and related procedures of dPEG biotinylation for related content, if you are interested in Molecular biology viscous specific dPEG biotinylation, Biostability high solubility solution dPEG biotinylation, Peptides amorphous specific dPEG biotinylation, you can consult our website, our website is www.pu-kang.com.

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