Covalent immobilization of antibodies to protein G beads is a basic molecular biology method, although the beads present poor recovery results. Our aim was to reuse the immobilized antibody-protein G complex on a very small scale, therefore we optimized the crosslinking procedure to be used on the wells of a standard 96-well microplate. The method used involves the affinity binding of the antibody to the protein G surface, followed by the immobilization step using different crosslinking reagents, DMP and BS³, quenching the crosslinking reaction, and binding the antibody-specific antigen. By scaling down the procedure, we were able to reuse the anti-EGFR crosslinked wells more than 20 times. This method can be used to perform assays on a wide range of solid supports containing the protein G in an immobilized form, including functionalized nanosensors, for immunoprecipitation, protein and cell lysate purification, target protein enrichment.

将抗体共价固定于蛋白G珠是一种基本的分子生物学方法，尽管珠的回收率很低。我们的目标是在很小的规模上重复使用固定的抗体-蛋白质G复合物，因此我们优化了在标准96孔微孔板的孔中使用的交联程序。所用方法包括抗体与蛋白G表面的亲和结合，然后使用不同的交联剂DMP和BS ^3进行固定化步骤，淬灭交联反应，并结合抗体特异性抗原。通过缩减程序，我们能够重复使用抗EGFR交联孔超过20次。该方法可用于对多种固定化形式的包含蛋白质G的固体支持物（包括功能化的纳米传感器）进行测定，以进行免疫沉淀，蛋白质和细胞裂解物纯化，目标蛋白质富集。