蛋白質純化技術

970603

授課教師 :
海洋生物技術系
蔡志明
 Electrophoretic methods

Simple electrophoresis describes the
situation in which the protein
molecules are separated in an
electric field at a constant pH and
current
簡單的描述電泳，就是在電場利用固定的 pH
值和電流使蛋白質分子分離。
 Isoelectric focusing 等電聚焦


Electrophoretic steps are not often
employed in protein purification 電泳
步驟在蛋白質純化不是經常利用的。
 Analysis for purity
 Electrophoretic analysis 電流生理學
 Resolution of separate components of a protein
mixture is achieved most clearly using an
electrophoresis method
 利用電泳方法，可以清楚得到蛋白質的組成分析。

Preparative electrophoresis has problems in the
design and use of apparatus, so despite its
obvious potential, it is not often used
 儘管電泳方法非常有用，但麻煩在機器的設計與用途，因
此不常使用。
 But in an analytical mode, electrophoresis is a
most widely method 但是在分析的方法中，電泳是最
普遍的。
 It is almost obligatory to characterize a purified
protein preparation by an electrophoretic
technique

它的特性是利用電泳技術來純化蛋白質。

Gel system 凝膠系統
 Before gel system were developed,
electrophoretic analysis was carried out in the
Tiselius free-boundary apparatus, requiring tens
of milligrams of proteins 在發展凝膠系統前，電泳分
析經過 Tiselius free-boundary 儀器，需要數十毫克的
蛋白質 。

Starch gel 澱粉凝膠

Synthetic gel medium- crossed linked
polyacrylamide 合成凝膠 - 經過聚丙烯
酰胺連結
 Polyacrylamide gel electrophoresis
聚丙烯酰胺凝膠電泳
 Simple (native) gel electrophoreis
簡單的凝膠電泳

 This involves running the sample in a

buffer at a pH where the proteins
remain stable and in their native
form 在 buffer 跑樣本的 pH 值需維持在
穩定且為原本蛋白質的 pH 濃度。
 This method was the original
procedure, making use both of
differences in charges between
proteins, and their different sizes
這種傳統的方法，是利用蛋白質之間電荷

The concentration of the
polyacrylamide can be adjusted over
a wide range according to the size of
molecules being separated 聚丙烯酰胺
的濃度可以根據分離分子的大小調整範圍
。

Acrylamide and bisacrylamide 丙烯酸
樹脂和雙丙烯醯胺
 Polymerization or gelation 聚合或凝膠
 Ammonium persulfate and TEMED 硫
酸銨和 temed
 Urea gels 尿素凝膠
 This second method is particularly
useful for proteins that are insoluble
at the low ionic strength needed in
electrophoresis
 蛋白質在低離子濃度是不易溶解的，這是
電泳第二個特別有用的地方。

Each protein is separated according
to charge and to subunit size 每一個
蛋白是根據電力與大小分離。
 SDS gels
 It involves running the electrophoresis after denaturing the
proteins with the detergent sodium dodecyl sulfate 變性的蛋
白質與洗滌劑十二烷基硫酸鈉涉及到電泳的進行。
 PAGE-SDS (polyacrylamide gel electrophoresis in sodium
dodecyl sulfate)

聚丙烯酰胺凝膠電泳在十二烷基硫酸鈉

Although separation is only on the
basis of size, resolution of closely
similar components is excellent 雖然
分離只能用在基本的大小規模，但是分析
類似成分的能力卻是優秀的。

The charge/size ratio is virtually
identically for all proteins, and
separation can occur only as a result
of the molecular sieving through the
pores of the gel 所有蛋白質的電荷與大
小比例幾乎是相同的，分離作用會在分子
篩選通過凝膠的孔隙時發生。
 Gradient gel
 Native protein 天然蛋白質
 It is run until no further movement occurs,
and a “focusing” or concentration of
diffuse protein zones can occur, resulting
in very fine resolution 直到沒有一個“重點”
或散開的蛋白區濃度可以持續運轉時，會產生了
極微細的分析。
 Gradient-SDS gel 有梯度的 SDS 凝膠
 Isoelectric focusing
 Isoelectric focusing involves setting
up a pH gradient and allowing the
proteins to migrate in an electric field
to the point in the system where the
pH equals their isoelectric point
 在 pH 值等於其等電點時，等電聚焦成立一
個 pH 梯度，並允許蛋白質遷移在一電場點
。
Immobilized pH gradient, IPG
固定化 pH 梯度
Two-dimensional systems
兩維系統
 Staining and detection of proteins after
electrophoresis
染色和檢測的蛋白質電泳

Coomassie brilliant blue Silver staining