Harrison Gerald, Haffey Patrick, Golub Ellis E
Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104, USA.
Anal Biochem. 2008 Sep 1;380(1):1-4. doi: 10.1016/j.ab.2008.05.009. Epub 2008 May 17.
We have developed a nanogram-level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein-gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10-20 mul) containing 1 to 5 mug protein. A small volume (2 mul) of protein solution is applied to nitrocellulose paper in a grid array and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (2-16 h), rinsed with water, and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than do conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples and compared with the Pierce Micro BCA Protein Assay Kit. In addition, the Golddots assay is evaluated with several known proteins with different physical properties.
我们基于胶体金与附着在硝酸纤维素纸上的蛋白质的结合，开发了一种纳克级定量蛋白质测定法。蛋白质 - 金复合物产生与存在的蛋白质量成比例的紫色，并且通过光密度测定法定量染色强度。典型的测定需要最少的起始材料（10 - 20微升），其中含有1至5微克蛋白质。将少量（2微升）蛋白质溶液以网格阵列形式施加到硝酸纤维素纸上并干燥。将硝酸纤维素纸在胶体金悬浮液中轻轻搅拌孵育（2 - 16小时），用水冲洗并扫描。通过对扫描图像进行光密度分析，与放置在同一硝酸纤维素网格上的蛋白质标准品进行比较，可以定量未知样品的蛋白质浓度。该测定法所需的样品量比传统蛋白质测定法少得多。在本报告中，对Golddots测定法进行了针对称重蛋白质样品的校准，并与Pierce Micro BCA蛋白质测定试剂盒进行了比较。此外，还使用几种具有不同物理性质的已知蛋白质对Golddots测定法进行了评估。
