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| 題 名 | 電泳膠片上果膠酯酶之快速活性染色法=A Rapid Method for Pectinesterase Activity Staining |
|---|---|
| 作 者 | 江啟銘; 侯文琪; 張為憲; | 書刊名 | 食品科學 |
| 卷 期 | 25:1 1998.02[民87.02] |
| 頁 次 | 頁46-58 |
| 分類號 | 341.91 |
| 關鍵詞 | 果膠酯酶; 果膠; 酯化度; 活性染色; 聚丙烯醯胺膠體電泳; Pectinesterase; PE; Pectin; Degree of esterification; DE; Activity staining; Polyacrylamide gel electrophoresis; PAGE; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | 以酸性膠體電泳進行高等電點果膠酯?pectinesterase; PE) (如高等植物PE)之分離，或以鹼性膠體電泳進行低等電點 PE(如愛玉子PE或微生物PE)之分離後，將電泳膠片與含有反應基質(1%高酯化度果膠，0.2M 氯化鈉及 pH 6.5 之 0.04M 磷酸鹽緩衝液)之聚丙烯醯胺膠片疊合後，依酵素活性高低靜置反應 10 秒至 5 分鐘，然後將含果膠之膠片立即浸於 0.05%ruthenium red 溶液中進行染色，即可在膠片上觀察到具深玫瑰紅色之PE活性區帶，以蒸餾水進行背景脫色後，可以作成乾片長期保存。當PE作用於高酯化度果膠使成較低酯化度果膠後，與 ruthenium red 結合量增加，使PE活性區帶呈現深玫瑰紅色，而膠片之背景則呈現極淡的粉紅色。所使用之果膠以酯化度 93％為最佳，而隨著酯化度之降低，活性染色效果也降低，在酯化度低於 65％時，則因背景顏色加深而使PE活性區帶難於辨別。 本 PE 活性染色法之優點包括:快速；容易觀察；可適用於高等電點及低等電點PE 之活性染色：操作簡易；失敗率低、再現性高、解析度高、靈敏度高；可重複活性染色(適合於不同活性PE同功?之偵測);不破壞電泳膠片，故可於活性染色後再進行CBR染色，免疫轉印及免疫染色、電溶離(electroelution)，乾片保存等操作。 |
| 英文摘要 | After separation of high pi pectinesterase (PE) isolated from higher plants by acidic gel electrophoresis, or after separation of low pi PE isolated from jelly fig or microorganisms by alkaline gel electrophoresis, the gel slice containing separated PE bands was put on the top of another polyacrylamide gel slice containing 1% high methoxy pectin, 0.2 M sodium chloride and 0.04 M phosphate buffer at pH 6.5 for enzyme reaction. After reaction at room temperature for 10 sec to 5 min depending on the PE activity, the gel slice containing pectin was immersed in 0.05% ruthenium red solution for staining, and after destaining with distilled water, the gel slice could be dried for long term preservation. After staining, a deep red color appeared at the locations of PE activity bands due to the reaction between ruthenium red and the free carboxyl groups released by PE reaction while the background was stained a weak pink color, depending on the degree of esterification (DE) of the pectin used. The best staining was achieved by using a pectin of 93% DE while the efficiency of staining became worse as DE was reduced, and the background and PE activity bands on the gel slice finally became indistinguishable when a pectin of DE lower then 65% was used. The advantages of this PE activity staining method are that it is rapid, easy to perform, easy to observe, and applicable to high pi and low pi PE's while it can be stained repeatedly, it can be applied to analyze PE isozyme, and there no damage of the clectrophoresis gel slice during the operation. Thus, it can be further used for CBR staining, immunoblotting, immunostaining, electroelution, or dehydration for preservation. |
本系統中英文摘要資訊取自各篇刊載內容。