CAL BIOCHEMISTRY 72 248 254 1976 apid and Sensitive Method

Last updated: 9/3/2023

CAL BIOCHEMISTRY 72 248 254 1976 apid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Dye Binding MARION M BRADFORD Reproduction Research Laboratories Department of Biochemistry University of Georgia Athens Georgia 30602 Received September 11 1975 accepted January 29 1976 A protein determination method which involves the binding of Coomassie rilliant Blue G 250 to protein is described The binding of the dye to protein auses a shift in the absorption maximum of the dye from 465 to 595 nm and is the increase in absorption at 595 nm which is monitored This assay is ery reproducible and rapid with the dye binding process virtually complete in pproximately 2 min with good color stability for 1 hr There is little or no nterference from cations such as sodium or potassium nor from carbohydrates uch as sucrose A small amount of color is developed in the presence of trongly alkaline buffering agents but the assay may be run accurately by the use of proper buffer controls The only components found to give excessive nterfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate Triton X 100 and commercial glassware detergents Interference by small amounts of detergent may be eliminated by the use of proper controls aboratory practice in protein purification often requires a rapid an sitive method for the quantitation of protein Methods presentl inal primary literature article that describes the Bradford Assay A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Then answer the questions below about the article 3 9 pts According to the article Coomassie Brilliant Blue G 250 can be red or blue a What causes the color change from red to blue b Approximately how long does it take for the color to change In the pre lab video instructions stated to incubate Bradford dye protein for 10 minutes According to Figure 3 in the paper how much would the absorbance change if I waited one hour to make my measurement 4 5 pts In the manuscript the author looks at a variety of substances to see if they interfere with the Bradford assay We will be using NaCl in our Buffer this semester Would 50 mM NaCl interfere with the assay at 595 nm Provide evidence for your answer