in an unknown sample by comparing its assay response to one which is already known. This has uses in a broad range of research topics which include protein analysis. Two bovine serum albumin (BSA) dilutions of unknown concentrations using the Bradford assay method of protein quantification were used. BSA is a fatty acid carrier protein in the blood; this is used as it stabilizes enzymes and has advantageous properties such as protection from oxidative damage and stabilization of proteins for analysis
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Proteins are large biological molecules that are made up of one or more chains of amino acids. Proteins perform many different functions within a living organism‚ such as catalysing metabolic reactions‚ replicating DNA‚ responding to stimuli‚ and transporting molecules from one location from another. All proteins differ from each other primarily by their sequence of amino acids which is usually determined by a nucleotide sequence of their genes‚ resulting in a three-dimensional fold that determines
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Objectives 1 1. To prepare a standard curve of absorbance versus protein concentration by using Bovine Serum Albumin (BSA). 2. To determine BSA concentration in two sample solutions. 3. To determine protein concentration in apple juice. Introduction What is Bradford protein assay? The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution [1]. There are several reagents that can be used to determine the concentration
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reagent‚ and ultraviolet light in order to determine the identity of six unknown solutions and the concentration of a bovine serum albumin sample. We were given three samples that lacked protein‚ and three samples containing proteins‚ and using a spectrophotometer we assessed the amount of light absorbed versus the light transmitted‚ based on the principles of the Beer-Lambert Law. The three proteins used included lysozyme‚ protamine sulfate‚ and bovine serum albumin‚ and the three non-protein samples
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Protein Assay. The main purpose of this experiment was to observe and record the various protein samples’ absorbency values through the calibrated readings of a spectrophotometer (595 nm calibration). Using the standard curve equation (y = 1.6147x + 0.0968) derived from determining the regression equation of different protein concentrations of BSA‚ an accurate form of protein concentrations were obtained for three different given samples‚ including an unknown. A notable empirical observation found in
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BRADFORD ASSAY Calculation Formulas: Absorbance of BSA = A595 nm Raw Data = Average of A595 nm of three wells ÷ 3 Example: A1+A2+A3 ÷ 3 In this case‚ 0.365+0.374+0.453 ÷ 3 = .397 Corrected Data =( (A595 nm well) – BackGround) or (Raw Data – Background) Background = negative control = Bradford Reagent + No Protein Background of this standard curve = A595 nm of well ‘A’ = .397 We used well ‘A’ as our negative control. DATA RAW DATA
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Winny Stephanie Experiment 1: Quantitative Determination of Protein Concentration Using the Biuret Test Table 1: Experimental protocol for construction of the protein standard curve Tube 1 2 3 4 5 6 Buffer (ml) 1.0 0.8 0.6 0.4 0.2 0.0 BSA Protein solution (10 mg ml-1) (ml) 0.0 0.2 0.4 0.6 0.8 1.0 Biuret reagent (ml) 4.0 4.0 4.0 4.0 4.0 4.0 Total Volume (ml) 5.0 5.0 5.0 5.0 5.0 5.0 Final protein concentration (mg ml-1) 0 2 4 6 8 10 Absorbance 0.000 0.092 0.163 0.272 0.363 0.474 Table
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curve with the understanding that the results on the unknown protein are semi-quantitative. Since most proteins are not available in large quantities‚ standard curves for protein assays are typically based on the use of either bovine serum albumin (BSA) or bovine gamma globulin (IgG). The Bradford protein assay is a method to determine protein concentration in the mixture. This method is based on the proportional binding of the dye Coomassie to proteins. If there is a higher protein content‚ the
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Figure1: Standard curve. This graph was constructed from the fixed amounts of Bovine Serum Albumin (BSA). Varying amounts of BSA at a concentration of .5µ/µl were mixed with the water and 1mL of coomassie dye. The equation y=0.0259x +0.0511 represents the best fit line for a data set taken from µg of protein equals two to ten µg. Optical density (OD) was surveyed using a Mach V visual spectrophotometer at 595. Worktable for Coomassie Assay Tube # | Sample ID | Water (µl) | Sample (µl) | Protein
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Procedure 4 Results 5-6 Discussion 7-8 Conclusion 9 References 9 Introduction The concentration of protein in a sample can be obtained by using the Bradford protein assay method. The method requires a spectrophotometer and it is easily operated. The Bradford protein assay is a colorimetic assay based on the interaction of the blue dye “Coomassie” and the protein. The blue ye firsts donates free electron to the protein and causes the protein to lose its native
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