When clear pellet comes mix this with mili Q water and store it at -20 Protein concentration of samples were determined using Bradford assay (Bradford,1976) and Lowry assay
(Lowry et al.
The Lowry assay
, which measures certain amino acids (6), and the Biuret assay on six control urines gave protein concentrations of 2734 [+ or -] 886 and 1915 [+ or -] 1132 mg/L, respectively.
Under denaturing and reducing conditions, 5-10 [micro]g of total protein determined by Lowry assay
(10) was separated on 12% sodium dodecyl sulfatepolyacrylamide gels as described previously (11).
After centrifugation (10 000g for 5 min), the protein content of the supernate (UPL stock solution, 1.9 g/L) was determined by Lowry assay
(32), and the UPL was diluted (in saline/sodium azide) to give 300 mg/L and 500 mg/L UPL protein calibrators, which were stored at -20[degrees]C in 100-[micro]L aliquots.
It is true that high urea concentration will increase absorbance reading in the Lowry assay
in the absence of any protein, but the effect of this on the calculated protein concentration can be reduced or eliminated.
However, many substances commonly used during protein purification interfere with the Lowry assay
. To overcome this, the BCA method was developed.