Bradford Protein Assay
An Le
Foundation Biology lab
BRADFORD PROTEIN DISCUSSION ESSAY
The appearance of blue color showed the present of protein in the BSA dilutions. The more diluted the solution was, the less blue it was. The R2 value of the standard curve of BSA dilution was obtained to be 0.9972, which is close to 1. The closer to 1 the R2 value was, the more accurate the linear portion was. The error percentage of each unknown was large: 25.9% for skim milk, 95% for soy sauce, and 64.7% for egg white. The vast difference between the theoretical protein concentration and experimental protein concentration of the unknowns showed that Bradford Protein Assay must have limitations. The Coomassie dye only interacts with certain amino acids such as: arginine, histidine, lysine, tyrosine, tryptophan and phenylalanine. However, each amino acid has different structure from each other; therefore the Coomassie dye will interact differently with each amino acid. The Coomassie dye molecules are bound to proteins by elctronstatic attraction enhanced by hydrophobic bonding (Tal et al. 1984). Besides the interaction between Coomassie dye and amino acids, some compounds can interfere the result of the Bradford assay such as: salt, fat, and detergent. Another factor that could influence on the Bradford assay is the protein sample must fall within the linear range of standard curve. Another possible explanation for the difference between theoretical protein concentration and experimental protein concentration is human factor. The bottom part of the cuvettes was not supposed to be touched by because that was a region in which the beam of light goes through. The absorbance values at 595 nm are part of the variable of the standard formula that was used to calculate the experimental protein concentration of the unknowns. Therefore, the adjustments in these absorbance values would effectively affect on the experiment protein concentration values. Pipetting could be another error source because it
Foundation Biology lab
BRADFORD PROTEIN DISCUSSION ESSAY
The appearance of blue color showed the present of protein in the BSA dilutions. The more diluted the solution was, the less blue it was. The R2 value of the standard curve of BSA dilution was obtained to be 0.9972, which is close to 1. The closer to 1 the R2 value was, the more accurate the linear portion was. The error percentage of each unknown was large: 25.9% for skim milk, 95% for soy sauce, and 64.7% for egg white. The vast difference between the theoretical protein concentration and experimental protein concentration of the unknowns showed that Bradford Protein Assay must have limitations. The Coomassie dye only interacts with certain amino acids such as: arginine, histidine, lysine, tyrosine, tryptophan and phenylalanine. However, each amino acid has different structure from each other; therefore the Coomassie dye will interact differently with each amino acid. The Coomassie dye molecules are bound to proteins by elctronstatic attraction enhanced by hydrophobic bonding (Tal et al. 1984). Besides the interaction between Coomassie dye and amino acids, some compounds can interfere the result of the Bradford assay such as: salt, fat, and detergent. Another factor that could influence on the Bradford assay is the protein sample must fall within the linear range of standard curve. Another possible explanation for the difference between theoretical protein concentration and experimental protein concentration is human factor. The bottom part of the cuvettes was not supposed to be touched by because that was a region in which the beam of light goes through. The absorbance values at 595 nm are part of the variable of the standard formula that was used to calculate the experimental protein concentration of the unknowns. Therefore, the adjustments in these absorbance values would effectively affect on the experiment protein concentration values. Pipetting could be another error source because it