Whole protein, protein concentration was estimated by Micro BCATM protein assay kit (Thermo scientific 23235) using bovine serum albumin as standard (0-40 µg/ml. BSA). The absorbance at 562 nm. (OD562) was measured and the protein concentration
was calculated using the standard curve which plotted between OD562 and BSA concentration on Y-axis and X-axis, respectively. Diluted sample by deionized water were mix with 500 l. freshly prepared Micro BCA working reagent(WR) by mixing 25 parts of
Micro BCA Reagent MA and 24 parts Reagent MB with 1 part of Reagent MC
(25:24:1, Reagent MA:MB:MC). When reagent MC is initially added to Reagent MA and MB, turbidity occurs that quickly disappears upon mixing to yield a clear-green solution. Pipette 100 µl of each standard and unknown sample replication into a microplatewell(Thermo scientificNo. 15041). Add 100 µl. of the WR to each well and plate thoroughly on plate shaker for 30 seconds then incubate at 37 C for 2 hours.
The absorbance at 562 nm was measured using a microplate reader