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Determinations Of The Protein Concentration Of A Cell Extract Using The 'bradford Method'

683 words - 3 pages

DETERMINATIONS OF THE PROTEIN CONCENTRATION OF A CELL EXTRACT USING THE 'BRADFORD METHOD'.CBDP21LFS0028PRACTICAL GROUP E14TH OCTOBER 2008ABSTRACTThe aim of the experiment is to find out the concentration of cell extracts (Bovine Serum Albumin, BSA) using the Bradford method. Stock solution of BSA was made by diluting the protein solution to prepare concentrations ranging from 0.0mg/cm3 to 1.0mg/cm3 (Peck, 1998). 5cm3 of the Bradford dye reagent was added to 100μl of the solution and allowed to stand for 28mins. The absorbance of each solution is measured at 595 nm with the following results: 0.00 mg/cm3 = 0.00, 0.2 mg/ cm3 = 0.21, 0.4 mg/ cm3 = 0.34, 0.6 mg/ cm3 = 0.56, 0.8 mg/ cm3 = 0.68, and 1.0 mg/ cm3 = 0.80. The values of the absorbance were plotted against protein concentrations and the resulting line was used to calculate the concentrations of the unknown samples and the aim was achieved.INTRODUCTIONProtein concentration determination of samples is very necessary and it's often used i.e. to know the amount of protein available in a sample. Various methods for determining the protein concentrations in a sample are available but vary in ease of use, cost, and sensitivity. The most commonly used methods are the Bradford assay, Lowry assay and the BCA assay. In this case, the Bradford method, which is a dye binding method, is used. It is faster, cheaper, and more sensitive, does not require heating, and has fewer mixing steps than the other methods above.This method is based on the binding of protein to a dye, leading to a shift in the absorbance maximum of the dye used. The dye, Coomassie blue, has a maximum of 464nm but when it binds to a protein, the maximum shifts to 595nm. After creating a standard curve of protein concentrations of the known samples against absorbance, the concentrations of the unknown can be calculated. [Holme and Peck, 1998]MATERIALS AND METHODSThe materials/methods used were as described in the practical schedule with the following details.The absorbance of each standard was measured at 595nm against a blank that was composed of 100μl of water and 5cm3 of Bradford reagent and was zeroed.Incubation period 28minsMeasurements were made on a spectrophotometer that had been warmed up for at least 30mins prior to measurements.Protein...

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