Protein assay

Cells were washed with DPBS without calcium or magnesium and lysed for 15–20 mins at 4°C by the addition of 350 μl of T-PER lysis buffer containing a protease inhibitor cocktail. The lysate was transferred to clean microcentrifuge tubes and stored at -80°C for subsequent analysis. Total protein was quantified using the Bio-Rad protein assay using bovine gamma globulin (IgG) as a standard. The lysis buffer was tested for possible interference with protein quantification and ELISA assays through the generation of standard curves with varying concentrations of T-PER lysis buffer. The concentration 0.1X T-PER was determined to be compatible with the Bio-Rad protein assay.

Total protein was extracted from 500 mg of onion bulb tissue, which had been frozen at −80 °C, with 500 mL of PBS buffer and quantified using a protein assay (Bio-Rad) with bovine gammaglobulin (SIGM) as the standard. Alliinase, as the positive control, was purified from fresh garlic cloves. The purified alliinase was also quantified using a protein assay (Bio-Rad) with bovine gammaglobulin (SIGM) as the standard. Nine μg of the extracted total protein and 175 ng of the purified alliinase in Laemmli Sample Buffer (Bio-Rad) were loaded onto a 10% polyacrylamide gel and electrophoresed. The protein was stained by Coomassie Brilliant Blue (CBB) or electrically blotted onto a polyvinylidene difluoride membrane (Bio-Rad) using a Trans-Blot SD Cell (Bio-Rad) following the manufacturer’s instructions. The blot was blocked by 5% skim milk solution and incubated overnight with an alliinase polyclonal antibody raised in a rat. Excess antibody was removed by two 30 min washes in Tween-PBS buffer, and the membrane was incubated for 1 h with an anti-rat IgG solution. Again, excess antibody was removed by three 30 min washes in Tween-PBS buffer, and the alliinase protein was detected using the HRP Conjugate Substrate Kit (Bio-Rad) according to the manufacturer’s instructions. The membranes were imaged on a ChemiDoc XRS (Bio-Rad).

The protein concentration of the solutions was determined by the Bio-Rad Protein Assay using bovine serum albumin as a standard (Bio-Rad, CA, USA). The Bio-Rad Protein Assay is a dye-binding assay in which a differential color change of a dye occurs in response to various concentrations of protein.