Microplate dc protein assay

The protein concentration of lunasin-based commercial products and samples from ultrafiltration production of lunasin-enriched flour were determined by microplate DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA) as previously reported [1 (link)] and calculated using a bovine serum albumin (BSA) standard curve (y = 0.0003x + 0.0209, R² = 0.99).

Cell pellets were harvested from 50 mL of each culture and resuspended in 200 µL of protein extraction buffer (1X AEBSF, Roche and 1X PSB, Agrisera). Samples were then homogenized using the CY:24×2 rotor of the FastPrep-24 Instrument (MPBIO) for two 1 min periods with 1 min of cooling on ice after each treatment. The protein extracts were then centrifuged at 10,000×g for 3 min to remove insoluble material and unbroken cells. Protein concentrations of extracts were quantified using the microplate DC protein assay (Bio-Rad) with bovine gamma globulin (BGG) as a comparative protein standard.
The sample preparation, SDS-PAGE, immunoblotting and quantitation protocols were followed according to those described previously [26] (link). Immunoblotting of PSHCP required a shorter transfer of 45 min. onto nitrocellulose membranes. Primary antibodies (Agrisera) were diluted in 2% ECL Advance blocking agent (GE Healthcare) in TBS-T, RbcL 1∶10,000 and PSHCP 1∶2,000. HRP-conjugated goat anti-rabbit (Agrisera) was employed as the secondary antibody at a dilution of 1∶40,000. Immunoblots were developed with ECL Advance (GE Healthcare) and images were captured on a VersaDoc Imager (BioRad) and analyzed using Quantity One and Image Lab 3.0 software (BioRad).

A microplate DC protein assay (BioRad) was used to measure the protein contents of the samples. Each sample was analyzed in duplicate, and a pooled tissue sample was included in each plate to estimate the interassay coefficient of variation and the coefficient of variation, which was determined to be 4.9%. A standard bovine serum albumin (BSA) containing 0.2 mg/mL to 2.0 mg/mL protein was prepared in the same homogenizing buffer and analyzed along with samples. A fraction of 5 μL of each sample and standard protein was added to the well of a 96-well microplate followed by adding 25 μL of reagent A and 200 μL of reagent B as recommended by BioRad protocol. The plate was placed on the plate mixer and mixed for 5 sec, and then it was incubated at room temperature for 15 min. The absorbance at 750 nm was determined spectrophotometrically, the protein concentration of each homogenate was extrapolated from a standard curve. Samples of an extract of MCF7 cells and a protein extract from a control cell line were included in each run as positive controls.