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A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding

A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,Analytical Biochem

A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding   (Citations: 58610)
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering 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. Laboratory practice in protein purification often requires a rapid and sensitive method for the quantitation of protein. Methods presently available partially fulfill the requirement for this type of quantitation. The standard Lowry procedure (1) is subject to interference by com- pounds such as potassium ion (2), magnesium ion (3), EDTA (4), Tris (3), thiol reagents (2), and carbohydrates (5). The relatively insensitive biuret reaction (6) is subject to interference by Tris (7), ammonia (8), and glycerol (9). Even the modified procedure for eliminating problems with the Lowry and biuret assays (IO, 11) present problems since more complications and time are involved in the modified procedures. The dye binding techniques in the literature are for the most part insensitive assays involving the binding of Orange G to protein (12- 16). The excep- tion to this rule is the Amidoschwarz 10-B binding assay (17). This procedure, too, has its drawbacks since the precipitation of the protein by trichloroacetic acid followed by filtration on Millipore membranes is required. The protein assay herein described eliminates most of the problems involved in the procedures described above, and is easily utilized for
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