Methods for Determining Protein Concentration

Methods for Determining Protein Concentration - Methods for...

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Unformatted text preview: Methods for Determining Protein Concentration: Biuret, Spectrophotometric, and Lowry Assays Anna Krajec September 17, 2011 Lab Section 01 Biuret Experiment Standard BSA 6 mg/Ml Water [Protein] A 540 500 L 0.0 L 6.00 mg/mL .255 350 L 150 L 4.20 mg/mL .200 150 L 350 L 1.8 mg/mL .078 100 L 400 L 1.2 mg/mL .047 50 L 450 L 0.60 mg/mL .014 0 L 500 L 0.00 mg/mL .000 Biuret Protein Unknown (#7) 500 L 0.0 L .200 Determining the Unknown Spectrophotometric Assay 0.5 mg/mL (BSA) Water [Protein] A 280 0.0 mL 5.0 mL 0.0 mg/mL 1.0 mL 4.0 mL 0.1 mg/mL .0979 3.0 mL 2.0 mL 0.3 mg/mL .2869 5.0 mL 0.0 mL 0.5 mg/mL .3520 Spectrophotometric Unknown (#15) 5.0 mL 0.0 mL .1762 Determining the Unknown Lowry Assay Tube No 1.0 mg/mL BSA Water [Protein] A 595 1 600 L 400 L 0.60 mg/mL .630 2 500 L 500 L 0.50 mg/mL .540 3 400 L 600 L 0.40 mg/mL .469 4 300 L 700 L 0.30 mg/mL .362 5 200 L 800 L 0.20 mg/mL .251 6 100 L 900 L 0.10 mg/mL .242 7 0 L 1000 L 0.00 mg/mL .039 Lowry Unknown (#19) 8 500 L 500 L .095 9 1000 L 0 L .253 Determining the Unknown Questions 1. Which method was easiest? The Spectrophotometric Assay was the easiest. It had the least preparation time and fewest steps, meaning less room for error. It simply consisted of putting together the test tubes and running them through the spectrometer, which gave an exact reading and did not require any guessing. 2. Which method showed the best linearity? Why is linearity important? The Biuret Experiment showed the best linearity, with an R 2 value of 0.9918. Linearity is so important because the standard curve equation can then be applied to unknown concentrations with accuracy. If the points on the curve were all over the place and a lower R 2 value was calculated, the standard curve equation could not be trusted to truly determine the concentration of proteins that were unknown or not tested in that experiment. 3. Which method would be the best to use for a wide range of protein sources? Why? The Biuret method would be the best for use with a wide range of protein sources, since it only requires that the substance have peptide bonds, which all proteins do. It does not rely on the presence of aromatic amino acids like tyrosine and tryptophan like the Lowry and Spectrophotometric Assays do. Any proteins concentration could be identified with the Biuret method....
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This note was uploaded on 12/10/2011 for the course BCH 5045 taught by Professor Guy during the Fall '08 term at University of Florida.

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Methods for Determining Protein Concentration - Methods for...

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