electrophoresis and blotting 电泳与转渍技术.ppt

* * * Electrophoresis and Blotting 電泳與轉漬技術 大小、形狀、帶電荷 Argarose gel 洋菜瓊脂糖 電泳槽 RNA Protein 核酸, 蛋白質 電泳 轉漬技術 Argarose gel 洋菜瓊脂糖 SOUTHEPNBLOT南方轉漬法(南方墨點法) 1.螢光劑(如溴化乙錠EtBr)與DNA 結合成為肉眼可見的帶(band) Starch澱粉, agar洋菜, agarose洋菜瓊脂糖, or polyacrylamide聚丙烯醯胺. direct current 2. probes WHAT IS ELECTROPHORESIS?何謂電泳? Electrophoresis is the biochemical technique used for separating compounds in an electrical gradient電流梯度. The separation of compounds is based on variations in molecular or physical structure and chemical properties (e.g., size, shape, and natural charges).化合物的分離是依據分子或物理的結構、化學性質(大小、形狀、帶電荷) 的不同來進行 If significant natural differences do not occur, the experimental environment (e.g., pH) may be manipulated to effect separation. 物質本身特性不大可利用實驗環境變化(酸鹼值)達到分離目的 Migration is affected by charge density, molecular size and shape, medium characteristics, and buffer properties. 移動受到電荷密度、分子大小、形狀、介質特性和緩衝液性質有關 Support media may have sieving properties, retarding the mobility of large molecules while allowing small ones of identical charge density to move with relative ease. 介質有過篩性質，可延緩大分子移動速率，小分子則容易移動。 Molecules with large charge density will migrate faster than those with smaller charge densities, and some electrophoretic procedures (SDS) are able to cancel out the effect of charge so that molecules are separated on the basis of their molecular weight. 電荷密度大的分子移動快於電荷密度小的分子，但是一些步驟可抵償電荷效應，使分子能夠依照分子量不同而分離開來。 SUPPORT SYSTEMS Electrophoresis is commonly conducted in a matrix or support system, the common ones being gels prepared from starch澱粉, agar洋菜, agarose洋菜瓊脂糖, or polyacrylamide聚丙烯醯胺. Gels are prepared to give desirable physical properties to effect separation. 凝膠的物理性質可促進分子有效分離 Starch gelis relatively inexpensive; however, its weaknesses include low strength (fragile) and variability among sources that prevent the reproducibility of experimental results. 澱粉膠張力太差、易碎，也會因來源不同品質不定而使實驗結果誤差提高。 Agar is widel