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生命科學實驗

生命科學實驗. Immunological (Western) analysis of GST fusion protein. Day1: SDS-PAGE → Transfer . Day2: →Blocking → First antibody incubation → Wash → Secondary antibody incubation → Wash → Detection. Gel electrophoresis. Transfer. Blocking. Primary antibody. Secondary antibody.

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生命科學實驗

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  1. 生命科學實驗 Immunological (Western) analysis of GST fusion protein

  2. Day1: SDS-PAGE → Transfer Day2: →Blocking → First antibody incubation → Wash → Secondary antibody incubation → Wash → Detection

  3. Gel electrophoresis Transfer

  4. Blocking Primary antibody Secondary antibody Chemiluminescent detection (Colorimetric/ Radioactive/ Fluorescent)

  5. Immunological (Western) analysis http://www.youtube.com/watch?feature=player_embedded&v=VaXFwfkLdgw http://www.youtube.com/watch?feature=player_embedded&v=BNAeFlcCy3o http://www.youtube.com/watch?feature=player_embedded&v=TqQy3pJWsKM http://www.youtube.com/watch?feature=player_embedded&v=X9xPGVfdpEE http://www.youtube.com/watch?feature=player_detailpage&v=JMtvzyoapd0

  6. Immunogen (antigen) and Hapten 免疫原(抗原) 半抗原 Epitope Immunoglobulin = antibody 免疫球蛋白 抗體

  7. Immunological (Western) analysis

  8. Western Blot顯色的方法主要有以下幾種： Chemiluminescent detection (化學冷光法) Colorimetric detection (呈色法) Radioactive detection (放射自顯影) Fluorescent detection (底物螢光) 呈色法 ( Colormetirc/Chromogen )：由於直接顯色而操作簡便且成本低。最為大家熟悉的應當是 HRP（Horseradish Peroxidase，屬於過氧化物酶）和 AP（Alkaline Phosphatase），此外還有比較少見的葡萄糖氧化酶（Glucose Oxidase）、β-Galactosidase ( β半乳糖苷酶 )。 Horseradish Peroxidase (HRP) 是最常見的酶促發光或顯色的交聯酶，由於 HRP 活性高、特異性強、分子量小（40 kDa）且穩定及作用受質範圍廣的優點而得到廣泛使用。HRP 的受質種類不少，主要可以分為冷光和呈色兩大類。對於受質的選擇，主要考慮的是靈敏度、背景和使用的方便性和穩定性——比如受質的可溶性高，使用上就比較方便；對於西方墨點法來說產物不可溶有助於信號在原位的積累和結果的穩定（可溶性高的受質適合於 ELISA）。

  9. HRP 呈色受質比較：

  10. Expression of GST-LEU2 Protein GST GST-LEU2 GST-leu2 IPTG (h): 0 0 0 0 0 0 3 3 3 3 3 3 5 5 5 5 5 5 Green Angel Protein Staining 170 GST GST-LEU2 GST-leu2 130 IPTG (h): 95 72 170 55 130 43 95 WB: R anti-GST (1:3000) 34 72 26 55 17 43 34 26 17 20121108 SCH lab

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