Underwood P A, Bean P A, Mitchell S M, Whitelock J M
CSIRO Molecular Science, P.O. Box 184, N. Ryde, NSW 1670, Australia.
J Immunol Methods. 2001 Jan 1;247(1-2):217-24. doi: 10.1016/s0022-1759(00)00327-6.
Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.
血清是大多数体外细胞培养基的常见成分，原代细胞培养基尤其如此。对特定黏附分子或生长因子的细胞反应研究常常因血清补充剂中存在这些分子而受到干扰。我们描述了一种从血清中去除玻连蛋白和纤连蛋白的联合亲和方案。该方案也可用于纯化这些分子。我们还描述了使用肝素-琼脂糖去除促生长成分的方法。由于玻连蛋白和纤连蛋白都能与肝素结合，这些分子首先被去除，而肝素-琼脂糖去除步骤在最后进行。该方案以优化的形式提供了一个详细的逐步指南，以实现血清的定量去除，并提供了有关陷阱和问题的额外信息。它对希望准确确定细胞、细胞外基质分子和生长因子之间关系的人应该会有用。
