Roegener Jan, Lutter Petra, Reinhardt Ralf, Blüggel Martin, Meyer Helmut E, Anselmetti Dario
Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany.
Anal Chem. 2003 Jan 1;75(1):157-9. doi: 10.1021/ac020517o.
Visualization of proteins inside acrylamide and other gels usually relies on different staining methods. To omit the protein-staining procedure, we visualized unstained proteins inside acrylamide gels by laser excitation with ultraviolet (UV) light (280 nm, 35 mJ/cm2) and directly detected native UV fluorescence. In one-dimensional gels, a detection limit as low as 1 ng for bovine serum albumin and 5 ng for other proteins with a linear dynamic range (2.7 orders of magnitude) comparable to state of the art fluorescent dyes could be achieved. In addition, the application of this method to 20 microg of a whole cell lysate separated in a two-dimensional gel showed more than 600 spots. Since protein labeling always represents a serious obstacle in protein identification technologies, the working efficiency with our procedure can be considered as a significant improvement for protein visualization and reproducibility in proteomics.
丙烯酰胺凝胶及其他凝胶内蛋白质的可视化通常依赖于不同的染色方法。为省去蛋白质染色步骤，我们通过用紫外光（280纳米，35毫焦/平方厘米）进行激光激发来可视化丙烯酰胺凝胶内未染色的蛋白质，并直接检测天然紫外荧光。在一维凝胶中，对于牛血清白蛋白，检测限低至1纳克，对于其他蛋白质则为5纳克，其线性动态范围（2.7个数量级）与现有最先进的荧光染料相当。此外，将该方法应用于在二维凝胶中分离的20微克全细胞裂解物时，显示出600多个斑点。由于蛋白质标记一直是蛋白质鉴定技术中的一个严重障碍，我们的方法的工作效率可被视为蛋白质组学中蛋白质可视化和重现性方面的显著改进。
