Calibration curve Lowry protein assay

A standard Lowry-based protein assay has been adjusted to the special conditions encountered with skin [126], Basically, proteins reduce an alkaline solution of Cu(II)-tartrate to Cu(I) in a concentration-dependent manner. Then, the formation of a blue complex between Folin-Ciocalteau reagent (a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids) and Cu(I) can be measured spectrophotometrically at 750 nm. A calibration curve can be obtained by dissolving known amounts of stratum corneum in 1 M sodium hydroxide. A piece of tape that has not been in contact with skin is subjected to an identical procedure and serves as negative control. The method was recently adapted to a 96-well plate format, notably reducing analysis times [129],... 

With reagents prepared in advance, the Lowry assay requires 1 h. A 400-pL sample is required, containing 2-100-pg protein (5-250 pg/mL). Nonlinear calibration curves are obtained, due to decomposition of the Folin reagent at alkaline pH following addition to the sample that results in incomplete reaction. Interferences include agents that acidify the solution, chelate copper, or cause reduction of cop-per(II). 

The Lowry, Smith, and Bradford assays provide an estimate, rather than an exact measure of total protein. Why What is the importance of the standard protein chosen for the construction of calibration curves ... 

Protein was determined by the method of Lowry, using a calibration curve which had been obtained with a TK solution standardized by the Biuret method. TPP was determined by radioactivity measurements. The transketolase assay was carried out in the presence and absence of added thiamine diphosphate/Mg++. A stimulation of 13 % of enzymatic activity was found when the coenzyme was added, indicating a loss of bound coenzyme upon gel filtration. Assuming a linear relationship between enzymatic activity and thiamine diphosphate molecules bound to apotransketolase the value found for thiamine diphosphate was corrected by these 13 %. 

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