Electrophoresis and Blotting
電泳與轉漬技術
Argarose gel 洋菜瓊脂糖
核酸, 蛋白質
RNA
Protein
電泳
大小、形狀、帶電荷
電泳槽
轉漬技術
Argarose gel 洋菜瓊脂糖
SOUTHEPNBLOT
南方轉漬法(南方
墨點法)
direct current
Starch澱粉, agar洋菜,
agarose洋菜瓊脂糖, or
polyacrylamide聚丙烯醯胺.
1.螢光劑(如溴化乙錠EtBr)與DNA
結合成為肉眼可見的帶(band)
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-PAGE) 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 widely used in microbial research because of its
versatility, strength, and clarity, among other properties.
 洋菜張力強度夠，透明度高，常用於細菌研究。
 Agarose is clearer, stronger, and even more versatile. Its large
pores suit the separation of large biomolecules like nucleic
acids.
 洋菜瓊脂糖透明度更高，張力更強，有較大的孔洞，適合大分子
(例如核酸)的分離。
 Polyacrylamide gels have more strength than agarose
and can be prepared to provide thin gel thickness and
a wide range of pore sizes. Preparation of
polyacrylamide is more complex, not to mention the
fact that unpolymerized polyacrylamide is toxic.
 聚丙烯醯胺 強度大於洋菜瓊脂糖，並且能提供較薄厚度和大範
圍孔洞的膠體面積，有毒!
 Agarose is routinely used in molecular biotechnology
大型垂直電泳槽
大型垂直電泳槽
Mupid-ex可以分析DNA．RNA及蛋
白質等
VISUALIZATION
顯像系統
 Once electrophoresis is completed, the
researcher needs to visualize the relative
locations of the separated molecules, which
usually occur as either bands or spots.
 當電泳分離完成，需將已分離的分子相對位置顯現出來，
通常呈現條狀或點狀。
 Because biomolecules (especially nucleic acids)
are often noncolored, scientists need additional
techniques to make these bands or spots visible.
Gel visualization techniques may be grouped into
two categories: direct treatment and use of probes.
Direct treatment直接處理
 In protein electrophoresis, gels are often exposed to dyes (including
color dyes) to stain these biomolecules. Dye staining is a quick and
inexpensive technique. 將膠體放置在染劑中，進行染色。
 However, it has significant setbacks, including significant
background noise. 染劑會殘留在膠體上使背景顏色加深。
 Furthermore,dyes tend to fade with time.
染劑隨時間會退色
 Among the dyes commonly used are Amido Black 10B, Ponceau S
Fast Green, and Coomassie Blue dyes (especially type R 250).
 In addition, nucleic acids can be stained with fluorescent dyes and
visualized under uv light. This technique requires special equipment.
核酸可利用螢光染劑
Use of probes使用探針
 Probes utilize matching sequences to select specific bands, thereby
reducing the number of bands visualized to a minimum.
探針是利用互補的原理來標定特定分子產生條狀顯影。
 Stains can be labeled in various ways: by antibodies, nucleic acids,
radioisotopes, or those that are enzyme-linked. Antibodies are best
used on blots but are prone to cross-reactions. Nucleic acids and
proteins require denaturing.
 染色方法可藉由抗體、核酸、放射線同位素、或是酵素連結方式。抗
體最常用於點漬，但易發生交叉反應，核酸與蛋白質都需要變性。
 Antibody labeling is based on immunological binding to specific
proteins (antigens).
 抗體是以免疫作用方式結合到樣本中特定蛋白質
 。
 Enzyme-linked probes require an appropriate substrate that is
converted into a visible product. 連接酵素作用則需要受質存在。
direct current
Starch澱粉, agar洋菜,
agarose洋菜瓊脂糖, or
polyacrylamide聚丙烯醯胺.
1.螢光劑(如溴化乙錠EtBr)與DNA
結合成為肉眼可見的帶(band)
2. probes
BLOTTING
轉漬法
 Blotting is the technique of transferring electrophoretic
products onto other materials prior to visualization. 將電
泳產物轉印到其他其他物質再進行分析。
 Two of the most common materials are nitrocellulose
and cellulose.常用材料有硝酸纖維素與纖維素兩種。
 Nitrocellulose is not stable in alkali, which is a significant
limitation to its use, since blotting methods tend to use
high pH buffers to minimize the tendency of singlestranded DNA molecules from reassociating and thereby
increasing their transfer to the blotting membrane. 硝酸
纖維素對鹼不穩定，而在轉漬過程中會加入鹼性溶液減少
單股DNA再結合，因此使用上明顯受限。
轉漬法
Nylon-66
Nylon-66 derivates provide the other
popular blotting membrane. Nylon is
several times stronger than nitrocellulose
and has a higher binding capacity,
allowing it to bind to nucleic acids in low
ionic strength buffers. it is also able to
handle more rounds of reprobing.
尼龍-66更耐用，可重複使用，有較佳的結
合力，可在低離子強度中結合核酸。
SOUTHEPNBLOT
南方轉漬法(南方
墨點法)
轉漬技術
Southern hybridization
Transfer buffer
SOUTHEPNBLOT
南方轉漬法(南方墨點法)
The steps in Southern blot are as follows:
 1. Cloned DNA is cut by one or more restriction
enzymes.
 以限制酶對於想要選殖的DNA進行切割
 2. Fragments are submitted to get
clectrophoresis for separation, then stained with
ethidium bromide (EtBr) for visualization and
photographed under ultraviolet light.
 切割的DNA進行電泳分離，以溴化乙錠染色並在紫
外線燈下觀察。
 3. DNA in the gel is denatured into single-stranded
fragments.膠體中DNA變性成單股
 4. Denatured DNA is transferred to DNA-binding
material (commonly, nitrocellulose or nylon). 變性後DNA
轉印到硝酸纖維膜上，轉印的過程稱為轉漬
 5. DNA in the filter is immobilized by baking at
80 ℃ or exposing to uv light to crosslink the
fragments to the membrane.
 轉印到膜上DNA以80℃烘烤或暴露在紫外光下，使
DNA與膜上成分交互作用增加固定力。
 6. The DNA is hybridized in situ with a
radioactive probe. This may be done in a
beatsealed food bag containing probe solution.
將固定好的轉印膜放入含有探針溶液的袋子中，
使DNA與探針進行雜交作用。
NORTHERN BLOT
北方轉漬法(北方墨點法)
Uses of Northern blot include:
 1. Studying patterns of gene expression in
embryonic and adult tissues.
 2. Detecting alternative splicing of mRNA and
multiple transcripts derived from a single gene.
 3. Characterizing and quantifying
transcriptional activity of a specific gene in
different cells, tissues, or organisms (the density
of RNA band on X-ray film relates to the amount
of RNA transcribed).
WESTERN BLOT
西方點漬法(西方墨點法)
 Western blot is used for protein analysis. 分析蛋白質的技術
 Protein identification is based on both antibody reactions and antigens.
抗原抗體的反應原理
 Proteins are separated by a denaturing SDS polyacrylamide gel.
Following electrophoresis, the proteins are transferred (blotted) to a
nylon membrane.先利用SDS聚丙烯醯胺電泳分離再轉印到尼龍膜上。
 The membrane is then exposed sequentially to solutions containing a
primary antibody, followed by a secondary antibody to which an
enzyme is coupled. 尼龍膜上依序加入一級抗體、二級抗體(帶有酵素的)
 All sites on the membrane that do not contain blotted protein from the
gel are nonspecifically ‘blocked’ so that antibody (serum) will not
nonspecifically bind to them to produce a false positive result.
膜上未有轉印蛋白的部位用非專一性的填充物填滿，抗體才不會發生非
專一性結合，也才不會造成結果出現假陽性。
 The membrane is then soaked in a substrate solution to develop the
color reaction, which results in identifying the antigen as a band. 將尼龍
膜至於充滿酵素受質的溶液中，受質經由二級抗體上的酵素催化作用，
使真正有抗原呈現的部份出現條帶。