Ghosh S, Gepstein S, Heikkila J J, Dumbroff E B
Department of Biology, University of Waterloo, Ontario, Canada.
Anal Biochem. 1988 Mar;169(2):227-33. doi: 10.1016/0003-2697(88)90278-3.
Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w/v, in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng.microliter-1, respectively.
使用考马斯亮蓝染料结合法并结合扫描密度测定法，可快速测定植物和动物组织粗提物中的蛋白质含量。使用1毫升提取缓冲液从100毫克新鲜冷冻或干燥研磨的组织中提取总蛋白质。将1微升标准溶液或粗提物等分试样成行点样在大小合适的Whatman 3MM层析纸上。干燥后的样品用考马斯亮蓝R - 250（在酸化的50%甲醇中，0.2%，w/v）染色20分钟，并用酸化的20%甲醇冲洗两次。干燥后，使用扫描密度计读取蛋白质浓度作为反射率，并使用数字积分仪记录峰高或峰面积。在另一种方法中，从样品纸上切下每个斑点，使用超声波清洗器在1%十二烷基硫酸钠（SDS）中洗脱染料 - 蛋白质复合物。随后使用酶联免疫吸附测定酶标仪在微孔样品架中于590纳米处读取吸光度。这两种方法都比先前报道的方法具有明显优势。当处理大量样品时，它们速度明显更快，可避免叶绿素、二硫苏糖醇、SDS、2 - 巯基乙醇、诺乃洗涤剂P - 40和苯甲基磺酰氟（以及其他蛋白酶抑制剂）的干扰，并且在灵敏度方面有显著提高，分别能测量低至100和200纳克·微升⁻¹的蛋白质浓度。
