2.6. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
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

Peanut kernels contain as much as 43%–52% fat. To reduce interference in subsequent experiments, the freeze-dried, powdered peanuts/peanut sprouts were defatted, and the protein was extracted. One gram of powder from each treatment was mixed with 10 ml acetone and stirred at 4 °C for 2 h. Mixtures were then centrifuged at 3000 g (TGL-10G, Shanghai Anting Scientific Instrument Factory Co., Ltd., China) for 10 min, and the precipitate was obtained. The precipitate typically required three to four rounds of degreasing until the supernatant was transparent. The precipitate was then naturally air dried in the fume hood.

SDS-PAGE was used to separate proteins extracted from the degreased powder using an SDS Kit obtained from Solarbio (Sinopharm Chemical Reagent Co., Ltd, Beijing, China). The SDS-PAGE method was modified from that described in Meng et al. [21] and used a Mini Protein Tetra System (BioRad, Hercules, CA, USA). Each electrophoresis sample (2.0 mg) was placed in a 1.5-mL centrifuge tube. Soluble protein extracts were mixed with an equal volume of sample buffer containing 1% β-mercaptoethanol. The mixture was boiled for 5–10 min for denaturation. Electrophoresis was performed on 13% acrylamide gels for 40 min at 80 V, followed by 3 h at 120 V. Protein marker standards were used to identify and estimate the major allergens and their molecular masses. To quantify the peanut major allergen Ara h 1, gels were scanned and analyzed with a Molecular Imager (AI600UV, GE Analytical Instruments, USA). The major allergens and their relative composition were calculated based on the band intensity and total area of their subunits.

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