Accordingto Kołodziejska, Sikorski and Niecikowska (1999), the Lowry method is based on two separate reactions. The first comprises formation of amide bonds with copper ion complex resulting into a reduced copper in alkaline solutions. Second is the reduction of Folin-Ciocalteu reagent by tryptophan and tyrosine residues. The reduced Folin-Ciocalteu reagent is blue in color and, therefore, detectable using a spectrophotometer ranging from 500-750 nm. However, the Biuret reaction is not highly sensitive and therefore use of Folin-Ciocalteu reagent makes the assay highly sensitive than a mere Biuret reaction (Nalinanon al.
2007). Nevertheless, the assay takes more time and is susceptible to many interfering compounds. Most of the interfering substances are commonly used as buffers in preparation of proteins (Lin & Liu 2006). Further, the Lowry assay is sensitive to variations in the quantities of tryptophan and tyrosine residues. According to Wang, Yang, and Du (2009), if the protein being assayed has got the unusual content of these compounds, a substitute should be used. Notably, the standard curve is linear in the 1 to 100 μ g protein region. The is commonly achieved between 500 to 750 nm.
Biuret technique: Protein determination through biuret technique is done using an ultraviolet wavelength of about 250-350nm (Aukkanit & Garnjanagoonchorn 2010). It shows the different spectrum with BSA and adsorption. Although in this experiment the maximum absorption was observed at 270, 280nm was preferred as the most appropriate wavelength. However, to obtain the highest sensitivity, the copper-iron concentration was required to be about 0.2% even though the NaOH concentration of less than 10% was the most desirable. Tartrate salt was used to the solubility of copper ion.
To arrive at different levels of concentration and absorption, the interpolation method was used. We determined the slope by taking two points along the straight line and calculate the change in y over the change in x.
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