Harake B, Caines P S, Thibert R J
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
J Clin Lab Anal. 1992;6(5):290-6. doi: 10.1002/jcla.1860060507.
Polymorphism of apolipoproteins AI and AII (apo AI and apo AII) can be easily investigated in plasma by a simple method involving a 30-min incubation of EDTA plasma in the presence of urea, dithiothreitol, and Nonidet P-40 followed by subsequent isoelectric focusing (IEF). The sample (2 microL) was applied to an ultrathin flat acrylamide gel of pH range 4-6, and focused using a Bio-Rad Mini IEF Cell for 1.5 h at a maximum of 500 V. Coomassie Blue R-250 was used to visualize the apolipoproteins. To verify the identity of the different apolipoproteins after IEF, the gel was immunofixed directly with anti-apo AI, or immunoblotted on polyvinylidene difluoride (PVDF) membrane using monospecific antibodies to apo AI and apo AII and an anti-immunoglobulin-alkaline phosphatase conjugate. High-density lipoprotein (HDL) was used as a standard for Apo AI variants. Employing these techniques, human plasma apo AI was resolved into one major band (apo AI0, pI 5.54), and four minor bands identified as apo AI+2 (pI 5.75), apo AI+1 (pI 5.66), apo AI-1 (pI 5.45), and apo AI-2 (pI 5.34). Apo AII was resolved into one major isoprotein designated as apo AII0 (pI 4.87), and two minor isoforms apo AII+1 and apo AII-1 which focused at pIs of 5.18 and 4.58, respectively. The results showed that these methods can be used to identify apo AI and AII isoforms without prior ultracentrifugation to isolate the HDL. The entire procedure, including IEF, fixation (chemical or immunofixation), and staining, can be accomplished in 5 h compared to 2 days using previously reported technique.(ABSTRACT TRUNCATED AT 250 WORDS)
载脂蛋白AI和AII（apo AI和apo AII）的多态性可通过一种简单方法在血浆中轻松研究，该方法包括在尿素、二硫苏糖醇和诺乃洗涤剂P - 40存在下将EDTA血浆孵育30分钟，随后进行等电聚焦（IEF）。将样品（2微升）加至pH范围为4 - 6的超薄扁平丙烯酰胺凝胶上，并使用Bio - Rad Mini IEF Cell在最大500伏电压下聚焦1.5小时。用考马斯亮蓝R - 250使载脂蛋白显色。为验证IEF后不同载脂蛋白的身份，凝胶直接用抗apo AI进行免疫固定，或使用apo AI和apo AII的单特异性抗体及抗免疫球蛋白 - 碱性磷酸酶偶联物在聚偏二氟乙烯（PVDF）膜上进行免疫印迹。高密度脂蛋白（HDL）用作Apo AI变体的标准品。采用这些技术，人血浆apo AI可分离为一条主要条带（apo AI0，pI 5.54），以及四条次要条带，分别鉴定为apo AI + 2（pI 5.75）、apo AI + 1（pI 5.66）、apo AI - 1（pI 5.45）和apo AI - 2（pI 5.34）。Apo AII可分离为一条主要的同型蛋白，命名为apo AII0（pI 4.87），以及两条次要的亚型apo AII + 1和apo AII - 1，其等电点分别为5.18和4.58。结果表明，这些方法可用于鉴定apo AI和AII的亚型，无需事先进行超速离心来分离HDL。整个过程，包括IEF、固定（化学固定或免疫固定）和染色，可在5小时内完成，而使用先前报道的技术则需要2天。（摘要截短于250字）
