Molecular Biology Problem Solver 36. 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. | Reagents Impure reagents from gel components to buffer salts stains and dyes can create problems similar to impure water. Gels will not be reproducible resolution may be poor and background staining may be substantial. For reproducible results and good resolution always use the purest components available electrophoresis grade. WHICH GEL SHOULD YOU USE SDS-PAGE NATIVE PAGE OR ISOELECTRIC FOCUSING The strategy you choose depends on your goal of course. If you want to determine the molecular weight of your protein use SDS-PAGE. If you want to measure the isoelectric point of your protein choose isoelectric focusing IEF . For proteomics work use 2-D electrophoresis IEF followed by SDS-PAGE . Native PAGE is used to assay enzyme activity or other biological activity for example during a purification procedure. Each kind of protein PAGE has issues to consider and these issues are addressed in the next section. Improving gel resolution is addressed in a separate section below. Will Your SDS Gel Accurately Indicate the Molecular Weight of Your Proteins Estimation of the molecular weight of the protein of interest accurate to within 2000 to 5000 daltons requires the protein band s to run within the middle two-thirds of the gel. This is illustrated in the graph of the log of the molecular weight of a set of standard proteins vs. the relative mobility of each one Figure . Note that the proteins with a relative mobility below or above fall off the linear portion of the curve. Thus the most accurate molecular weight values are obtained when the relative mobility of the protein of interest is between and . This means that if your protein doesn t enter the gel very well you must change the gel T before you can get a good molecular weight value. The sample may require a different better solubilization procedure also. See comments on sample preparation below. Should You Use a Straight Gel or a Gradient Gel If you want to resolve proteins that are within a few .
