Cheng Hao-Tsai, Hsieh Sen-Yung, Sung Chang-Mu, Pai Betty Chien-Jung, Liu Nai-Jen, Chen Carl P C
Division of Gastroenterology, Department of Internal Medicine, Linkou Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan.
Biomed Res Int. 2016;2016:5185317. doi: 10.1155/2016/5185317. Epub 2016 Feb 4.
Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established.
The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE) profiling quality, including number of protein spots and spot distribution.
The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin.
Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images.
胆汁是一种重要的体液，有助于脂肪消化以及内源性和外源性化合物的排泄。在本研究中，建立了一种改良的人胆汁样本制备方法。
该方法包括丙酮沉淀，随后使用市售的二维净化试剂盒进行蛋白质提取。通过二维电泳（2DE）图谱质量评估有效性，包括蛋白质斑点数量和斑点分布。
良性胆道疾病患者胆汁中的总蛋白为0.797±0.465μg/μL。与其他样本制备方法相比，先采用丙酮沉淀然后使用二维净化试剂盒进行蛋白质提取的样本制备方法在分辨率方面产生了质量更好的2DE凝胶图像。使用该方案，我们在凝胶图像上获得了约558个蛋白质斑点，且触珠蛋白、血清白蛋白、转铁蛋白和甲状腺素转运蛋白的蛋白质斑点呈现效果更好。
先采用丙酮沉淀然后使用二维净化试剂盒制备的胆汁蛋白质样本具有高蛋白质分辨率和显著的蛋白质图谱。这种优化的蛋白质制备方案可以有效浓缩胆汁蛋白质，去除丰富蛋白质和碎片，并在二维电泳凝胶图像上清晰呈现低丰度蛋白质及其异构体。
