The measurement of insoluble proteins using a modified Bradford assay

✍ Scribed by Simon M. Gotham; Peter J. Fryer; William R. Paterson

• Book ID: 102985040
• Publisher: Elsevier Science
• Year: 1988
• Tongue: English
• Weight: 511 KB
• Volume: 173
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(88)90199-6

✦ Synopsis

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, /Ilactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100°C to enable complete recovery of all insoluble proteins. B-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 pg, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay. o 1988 Academic PITSS, IN.