Kausch A P, Owen T P, Narayanswami S, Bruce B D
University of Connecticut, Storrs, USA.
Biotechniques. 1999 Feb;26(2):336-43. doi: 10.2144/99262rr04.
Currently, most organelle isolation procedures rely on physical parameters and centrifugation for separation. Here, we report the rapid and gentle isolation of a variety of organelles by immunolabeling whole cell lysates with organelle-specific antibodies and streptavidin magnetic particles followed by separation in a magnetic field. Using magnetic immunoabsorption, we have been able to specifically label mouse metaphase chromosomes and a variety of plant organelles, including: amyloplasts, choroplasts and nuclei from whole cell lysates of various plant tissues. We find that the distinct magnetic properties, surface characteristics and mean diameter-size ranges of different particle preparations significantly influence their specific utility for organelle isolations. By using an internal-field magnetic separation device, we have developed a method for quantitative recovery of labeled organelles in microarrays and tested a variety of antibodies to chloroplast outer envelope proteins for their ability to immune-isolate chloroplasts.
目前,大多数细胞器分离方法依赖于物理参数和离心分离。在此,我们报告了一种通过用细胞器特异性抗体和链霉亲和素磁性颗粒对全细胞裂解物进行免疫标记,然后在磁场中分离,从而快速、温和地分离多种细胞器的方法。利用磁性免疫吸附,我们能够从各种植物组织的全细胞裂解物中特异性标记小鼠中期染色体和多种植物细胞器,包括:造粉体、叶绿体和细胞核。我们发现,不同颗粒制剂独特的磁性特性、表面特征和平均直径大小范围显著影响它们在细胞器分离中的具体效用。通过使用内场磁分离装置,我们开发了一种在微阵列中定量回收标记细胞器的方法,并测试了多种针对叶绿体外膜蛋白的抗体免疫分离叶绿体的能力。