2.7. Extraction of peanut sprout proteins
This protocol is extracted from research article:
Effect of ultrasonic pre-treatment on Ara h 1 in peanut sprouts
Ultrason Sonochem, May 25, 2021; DOI: 10.1016/j.ultsonch.2021.105607

Degreasing was performed as described in Long et al. [15] with modifications. One gram of peanut or peanut sprout powder from each treatment was mixed with 10 ml of acetone, stirred at 4 °C for 2 h, and then centrifuged at 3000 g for 10 min to collect the precipitate. The above steps were repeated three times, and the precipitate was dried in the fume hood overnight.

Degreased peanut powder and protein extraction buffer (pH 8.0, 50 mM Tris HCl buffer) were mixed at a mass to volume ratio of 1:10, magnetically stirred at 4 °C for 2 h, and then centrifuged at 10,000 g and 4 °C for 30 min. The supernatant was collected and stored at −20 °C for future use.

Peanut protein extract was measured and precipitated with 80% and 100% ammonium sulfate as described previously [22]. SDS-PAGE electrophoresis was used to analyze the supernatant and the precipitated solution. Then 100% precipitated complex solution was placed into a dialysis bag with a molecular weight cutoff of 3.5 kDa for dialysis treatment. The dialysate was Tris HCl buffer (pH 8.0, 50 mM). After dialysis, the material was stored at −20 °C for further use.

Anion exchange chromatography of peanut sprouts was performed with an AKTA pure chromatography system (GE) and a DEAE Fast Flow column (GE Healthcare Company). The column was equilibrated until the baseline became smooth. Then a protein sample (5% of the column volume) was loaded onto the column. By continuously increasing the concentration of sodium chloride (dissolved in Tris-HCl as an elution buffer) to 0.4 M at a flow rate of 1.0 ml/min, the eluted proteins were monitored at 280 nm and then collected in the receptor machine. SDS-PAGE was subsequently performed to identify the components.

The purified protein samples were scanned with a UV spectrophotometer (TU-1810, Beijing Purkay General Instrument Co. Ltd., China) with a wavelength range of 200–450 nm and a spectral interval of 1 nm.

The purified Ara h 1 was scanned by infrared spectroscopy (TU-1810, Thermo Fisher Technology Co., Ltd, USA) to analyze changes in its secondary structure. The purified protein was diluted to 0.5 mg/ml and pressed flat into the sample chamber for spectral scanning. The scanning band was 4000–525 cm−1, the resolution was 4 cm−1, and the sample was scanned 16 times.

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