Molecular Biology Problem Solver 41. 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. | Background confined to the lanes is more likely to be related to non-specific antibody binding. Again be sure that you have optimized all your antibody concentrations. In order to pinpoint the problem it may be a good idea to run a control blot with no primary antibody. If bands show up in the absence of primary antibody the problem can be assigned to the secondary antibody in most cases the concentration of secondary antibody is simply too high. Otherwise your secondary antibody may have some specific affinity for something in your samples. If this is the case the only choice is to switch to a different secondary antibody or even a different detection approach . Protein A or biotin streptavidin . With other problems the guiding principle is still the same to try to glean as much information from the problem blot as possible to isolate each step in the process and change only one variable at a time. Holding each variable constant except for one makes each experiment decisive. This is the kind of situation in which detailed record-keeping is critical. When the performance of a system changes carefully going back over records often will suggest the source of the trouble. SETTING UP A NEW METHOD When setting up a new method it may appear that there is an impossible number of choices that need to be made all at once. Actually it s not so difficult. Your decision to go with another method should be based on the properties of your protein of interest the availability and nature of your samples your needs for reprobing or quantitation and the nature of your facilities. Read up on the relevant literature and at least in the beginning base your protocol on a published method. An important issue that needs to be addressed in setting up a new method is optimization of antibody concentrations. These concentrations will be different for every system. They can most easily be established through dot or slot blots the target protein either lysate or purified protein is spotted
