Molecular Biology Problem Solver 53. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | commercial vectors also include a significant number of extraneous residues derived from the polylinker region that could affect protein folding and activity. Thus while the one-size-fits-all commercial vectors are generally suitable for initial expression you will probably want to design more precise fusion constructs with either N-terminal or C-terminal tags. Tags can provide other benefits for expression. For example some researchers have found that the use of large soluble tags such as GST can enhance the solubility of certain proteins which favors the production of active protein Davies Jowett and Jones 1993 Weiss et al. 1995 Ciccaglione et al. 1998 . We have also observed that the additional C-terminal immunoglobulin Fc fusions sometimes result in the enhanced production of certain proteins secreted from CHO cells. Thus the addition of tags for detection and purification remains an empirical process as does the choice of a system in which to express the protein. Functional Assays In many cases the most efficient way to screen for expression is not through direct detection of the protein itself but through some kind of functional assay for the expressed protein s biological activity . apoptotic chemotactic proliferative or enzymatic . Crude cellular extracts or conditioned medium containing secreted proteins can sometimes be directly screened for biological activity. Functional assays are particularly useful when screening for the expression of a receptor whose ligand is known. In this case clones can be directly screened for cellular responses to added ligand. Calcium-mobilization and cAMP assays are two of the most commonly used methods of detecting signal transduction through G-coupled-protein receptors. TROUBLESHOOTING Finally after weeks or even months of selection you have isolated clonal cell lines that should be expressing large quantities of protein. However Western ELISA or functional assays are performed and they show that little or even no .
