Microtiter bradford protein assay

IPTG-induced E. coli JM109 containing modified MIT v2.1 plasmid for the acetylene reduction assay as described above were used to extract protein with 8 M urea and 2% SDS in 100 mM Tris-HCl pH 8.5. Protein extracts were stored at -80°C prior to processing. Protein concentrations were estimated using the Bio-Rad microtiter Bradford protein assay (California, USA) according to the instructions provided (Bio-Rad version: Lit 33 Rev C) and measurements were made at 595 nm using a SpectraMax Plus. Bovine serum albumin (BSA) standard was used in the linear range 0.05 mg/ml to approximately 0.5 mg/ml. The BSA concentration was determined by high sensitivity amino acid analysis at Australian Proteomics Analysis Facility (Sydney, Australia).

Proteins were extracted from three biological replicates of wholemeal flour (100 mg) in 1 mL of 8 M urea, 2% (w/v) dithiothreitol (DTT). The solution was thoroughly vortexed and sonicated for 5 min until completely mixed. Protein reduction continued on a thermomixer block, shaking at 1,000 rpm at 22°C for 45 min. The solutions were centrifuged for 15 min at 20,800 × g and the supernatants were used for subsequent analysis. Protein estimations were performed using the Bio-Rad microtiter Bradford protein assay (California, United States) following the manufacturer’s protocol. The protein extracts were diluted in water over two dilutions (1:20, 1:40) in duplicate and measurements were made at 595 nm using a SpectraMax Plus (Molecular Devices). Bovine serum albumin (BSA) standard was used in the linear range from 0.05 to 0.5 mg/mL. The BSA standard concentration was determined by high sensitivity amino acid analysis at the Australian Proteomics Analysis Facility (Sydney, Australia). Blank-corrected standard curves were run in duplicate. Linear regression was used to fit the standard curve. Protein sample wash and digestion steps were performed as precisely described in Colgrave et al. (2016) (link).