Freeman Lita A
Cardiovascular & Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2013;1027:369-85. doi: 10.1007/978-1-60327-369-5_18.
Western analysis of apolipoproteins, lipoproteins, and proteins involved in lipoprotein metabolism can be challenging due to their size, hydrophobic nature, and, in some cases, low abundance. Here we describe a Western blotting method that has been used successfully for many proteins involved in lipoprotein metabolism, as well as intact LDL or HDL particles. Proteins or lipoprotein particles separated by gel electrophoresis are transferred to a PVDF membrane in a Hoefer TE22 transfer tank with Tris-Glycine-SDS-Methanol transfer buffer. The membrane is blocked with 3 % BSA/5 % milk to prevent nonspecific binding of antibody to the membrane and is then incubated with primary antibody that binds specifically to the protein of interest. After washing away unbound primary antibody, the membrane is then incubated with an HRP-labeled secondary antibody that binds primary antibody. After washing away unbound secondary antibody, the membrane is then incubated with a substrate for HRP, generating a chemiluminescent signal at the location of the protein of interest. The protein is visualized by exposing the membrane to an autoradiography film or an imaging device. Information on the use of several human antibodies, including apoA-I, A-II, apoB, apoC-II, apoC-III, apoD, apoL1, apoM, PON1, SAA, ABCA1, nitrotyrosine, and LCAT, is provided. This method can be used for Western blotting of virtually any protein as well as native lipoprotein particles.
由于载脂蛋白、脂蛋白以及参与脂蛋白代谢的蛋白质的大小、疏水性，以及在某些情况下的低丰度，对它们进行蛋白质免疫印迹分析可能具有挑战性。在此，我们描述了一种蛋白质免疫印迹方法，该方法已成功用于许多参与脂蛋白代谢的蛋白质以及完整的低密度脂蛋白（LDL）或高密度脂蛋白（HDL）颗粒的检测。通过凝胶电泳分离的蛋白质或脂蛋白颗粒，在配备Tris-甘氨酸-SDS-甲醇转移缓冲液的Hoefer TE22转移槽中转移至聚偏二氟乙烯（PVDF）膜上。用3%牛血清白蛋白（BSA）/5%脱脂奶粉封闭膜，以防止抗体与膜的非特异性结合，然后与特异性结合目标蛋白质的一抗孵育。洗去未结合的一抗后，膜再与结合一抗的辣根过氧化物酶（HRP）标记的二抗孵育。洗去未结合的二抗后，膜再与HRP的底物孵育，在目标蛋白质所在位置产生化学发光信号。通过将膜暴露于放射自显影片或成像设备来观察蛋白质。文中还提供了几种人源抗体（包括载脂蛋白A-I、A-II、载脂蛋白B、载脂蛋白C-II、载脂蛋白C-III、载脂蛋白D、载脂蛋白L1、载脂蛋白M、对氧磷酶1、血清淀粉样蛋白A、ATP结合盒转运体A1、硝基酪氨酸和卵磷脂胆固醇酰基转移酶）的使用信息。该方法可用于几乎任何蛋白质以及天然脂蛋白颗粒的蛋白质免疫印迹分析。
