Quantification of erythrocyte membranes and cytoskeletons. The Lowry protein assay

Quantification of erythrocyte membranes and cytoskeletons. The Lowry protein assay 

Folin and Ciocalteu s reagent diluted 1 1 with water immediately before use 

Standard protein stock solution bovine serum albumin (4 g/1) in water 

Procedure The alkaline copper reagent was prepared just prior to use by mixing 1 vol. of copper sulphate solution with 1 vol. of sodium potassium tartrate solution followed by 98 vol. of the sodium carbon-ate/sodium hydroxide solution. Alkaline copper solution (5 ml) is added to 100 pA of sample, containing between 20-100 pig of protein, diluted with 900 pil of water. After 10 min 0.5 ml of diluted Folin and Ciocalteus reagent is added. The absorbance at 750 nm is read after 30 min and within 90 min from the final addition. 

Standard curve A series of dilutions of bovine serum albumin in water are prepared containing between 0 and 100 pig protein in 1 ml volume and the assay is performed as described above on each dilution. The exact concentration of the bovine serum albumin stock solution is determined using the molar absorption coefficient of 45 000 for bovine serum albumin at 279 nm after diluting to approximately 0.2 mg/ml. 

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