Microsoft PowerPoint - BCX2006 第三週蛋白質定量

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The obel Prize in Physiology or Medicine 2006 Goes to Professor Andrew Z.

Stanford University School of Medicine Stanford, A, USA Professor raig.

University of Massachusetts Medical School Worcester, MA, USA "for their

1 The obel Prize in Physiology or Medicine 2006 Goes to Professor Andrew Z. Fire, Ph.D. Stanford University School of Medicine Stanford, A, USA Professor raig. Mello, Ph.D. University of Massachusetts Medical School Worcester, MA, USA "for their discovery of A interference - gene silencing by double-stranded A" Ai 動畫 :

2 生化科技系 BST BX P1 生物化學實驗蛋白質定量

3 透析 (dialysis):desalting and exchange buffer At start of dialysis At equilibrium [igh salt] [Low salt] 1

4 α-d-1, 6-glucose Dextran α-d-1, 3-glucose Sephadex ross-linkage 2--2l Epichlorohydrin 2 Modified from Amersham Biosciences

5 膠體過濾法 :Separation, Desalting and Exchange buffer t = 5 min t = 10 min 緩衝液流洗方向 3

6 Trypsin (E ) 分子量 :23.3 kd (porcine) 分類 :Serine protease 作用方式 :Endopeptidase 專一性 :leaves proteins at the carboxyl side of lysine or arginine, except when either is followed by proline QGALFKVLGWYGAKPEM QGALFK V LGW YGAKPEM 4

7 M (chicken egg white ovomucoid) 含量 :11% of all egg white proteins 分子量 :28 kda, 186 a.a. (predicted MW: 20 kda) SDS-PAGE 之分子量 :34~49 kda arbohydrate content: 20~25% 醣基化 :5 個 -linked sugar chains kda M Stable to digestion and heat ooked eggs can cause allergic reactions in M-specific allergic patients 22 SDS-PAGE 5 Adapted from IAAI 2005; 136: 23-32

8 P1 蛋白質抽取 TA 沉澱第一次沉澱第二次沉澱成品檢定雞蛋上清透析液取蛋清丙酮沉澱丙酮沉澱 P3 膠體電泳法 TA 沉澱離心透析離心蛋白質定量離心取沉澱取沉澱 M 取上清沈澱回溶乾燥 P2 親和層析法

9 + - 蛋白質構造的骨架 : - group α α α α 3 + Peptide bond 6

10 蛋白質定量法 : UV absorbance Bradford dye-binding protein assay Biuret protein assay Lowry protein assay Bicinchoninic acid (BA) protein asay 7 Amido black protein assay used in criminal investigations to detect blood present with latent fingerprints o-phthalaldehyde (PA) protein assay

11 各種蛋白質定量法原理 : 8 Specific Binding Group + eme 206 nm (carbonyl) Peroxidase Trp Fe M Metal 280 nm (aromatic) UV Absorbance Phe Tyr Trp - Max. absorbance 260nm 275nm 280nm

12 Bradford Method: oomassie Brilliant Blue G-250 BG 是一種指示劑 470 nm 595 nm 加入蛋白質 9 酸性環境下呈茶色與蛋白質結合變藍色 Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,

13 各種蛋白質定量法原理 : 10 + Bradford dye-binding 206 nm (carbonyl) Trp 280 nm (aromatic) UV Absorbance S 3 - S 3 - S 3 - S 3 - -= S - 3 -= S Arg 2

14 蛋白質定量法 : 11 Biuret method Biuret 試劑乃鹼性硫酸銅溶液, 銅離子在鹼性溶液中, 會與蛋白質上的 peptide bonds 生成藍色或紫色的複合物, 由於 biuret 是在鹼性環境中和硫酸銅發生呈色反應之最小單位, 故稱此反應為 biuret 試驗測定 550 nm 的吸光 Lowry method (Folin-phenol 試劑法 ) 反應前半為 Biuret Method 二價銅離子在鹼性溶液中, 會與蛋白質 peptide bonds 上的 nitrogen 結合, 並還原為一價銅離子一價銅離子與 Tyr Trp 或 ys 會使 Folin reagent 內的 phosphomolybdic-phosphotungstic acid ( 磷鉬酸與磷鎢酸 ) 還原為藍色, 並在波長 660 nm 有很強的吸光此法比紫外光吸收法靈敏倍, 較 Biuret method 靈敏倍 Lowry et al. (1951). Protein measurement with the Folin-Phenol reagents. J. Biol. hem. 193: Bicinchoninic acid (BA) protein asay 亦是 Biuret 法的延伸在鹼性溶液中, 蛋白質使銅離子由二價還原成一價後, 進一步的以 BA 與一價銅離子結合產生很深的紫色, 在波長 562 nm 有很強的吸光本法的優點在於鹼性溶液中 BA 比 Folin reagent 穩定, 並且只需一個操作步驟 Smith et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85

15 蛋白質定量法 :Biuret method Urea 2 Biuret 2 2 u Protein u 2+ Peptide bond 12

16 蛋白質定量法 : Biuret method 加強版 Lowry method (Folin-phenol 試劑法 ) 反應前半為 Biuret Method 二價銅離子在鹼性溶液中, 會與蛋白質 peptide bonds 上的 nitrogen 結合, 並還原為一價銅離子一價銅離子與 Tyr Trp 或 ys 會使 Folin reagent 內的 phosphomolybdic-phosphotungstic acid ( 磷鉬酸與磷鎢酸 ) 還原為藍色, 並在波長 660 nm 有很強的吸光此法比紫外光吸收法靈敏倍, 較 Biuret method 靈敏倍 Lowry et al. (1951). Protein measurement with the Folin-Phenol reagents. J. Biol. hem. 193: Bicinchoninic acid (BA) protein asay 亦是 Biuret 法的延伸在鹼性溶液中, 蛋白質使銅離子由二價還原成一價後, 進一步的以 BA 與一價銅離子結合產生很深的紫色, 在波長 562 nm 有很強的吸光本法的優點在於鹼性溶液中 BA 比 Folin reagent 穩定, 並且只需一個操作步驟 Smith et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:

17 蛋白質定量法 :o-phthalaldehyde (PA) protein assay PA Primary amine Mercaptoethanol Excitation 340nm Emission 455nm 螢光 14

18 不同蛋白質定量法的測定值差異 Alcohol dehydrogenase α-amylase Bovine serum albumin arbonic anhydrase atalase α-hymotrypsin ytochrome β-galactosidase emoglobin (bovine) istones Lysozyme Myoglobin valbumin vomucoid Pepsin ibonuclease Trypsin inhibitor (soy) Transferrin Assay esults (protein mg/ml) Biuret Lowry Bradford Trypsin : Average 低估低估高估高估高估低估低估低估 Modified from Bio-ad

19 蛋白質定量法 : 不同標準品的標準曲線不同 Bradford dye-binding 非線性低估高估 16 Modified from Bio-ad

20 蛋白質定量法 : 不同標準品的標準曲線不同 BA Protein Assay IgG BSA A 562 nm 差異不大 Protein concentration (μg/ml) 17 Modified from Pierce

21 蛋白質定量法 : 溶液的適用性 eagents ompatible With Bio-ad Bradford Dye-Binding Protein Assay Acetate, 0.6 M Glycerol, 99% Acetone Glycine, 0.1 M Ammonium sulfate, 1 M EPES, 0.1 M Boric acid Kl, 1 M ATP, 1 mm Mercaptoethanol, 1 M DTT, 1 M Mgl 2, 1 M Eagle's MEM al, 5 M EDTA, 0.1 M Phenol, 5% EGTA, 0.05 M Phosphate, 1 M Ethanol SDS, 0.1% Formic acid, 1 M Sodium phosphate Fructose Tris, 2 M Glucose Triton X-100, 0.1% 18 Glutathione Urea, 6 M Derived from Bio-ad

22 各種蛋白質定量法的比較 : 定量方法靈敏度準確度其它說明 A 280nm mg 低高操作簡便樣本可回收其它物質干擾大樣品最好成分單一 A 205nm mg 低高操作簡便樣本可回收許多物質皆具有 carbonyl group Biuret mg 高呈色快速有腐蝕性銨離子干擾大 ( 如 Tris 硫酸銨 ) Lowry mg 中操作步驟多呈色形成較慢多種離子有干擾 BA mg 中高呈色較快生成物穩定 Bradford mg 中高立即呈色易受多種物質干擾短時間內可重複操作 19

23 P1 蛋白質抽取 TA 沉澱第一次沉澱第二次沉澱成品檢定雞蛋上清透析液取蛋清丙酮沉澱丙酮沉澱 P3 膠體電泳法 TA 沉澱離心透析離心蛋白質定量離心取沉澱取沉澱 M 取上清沈澱回溶乾燥 P2 親和層析法

材料與方法

材料與方法 10-12 250-350 Sprague-Dawley rat ( SD ) 12 ( AM 8 00-PM 8 00 ) (Control group) (HS) (Heat shock group) 15 0 4 6 16 24 (PAE) ( Pulmonary air embolism group) 20 40 (25 l/min) 40 17 20 (HS&PAE) 15 4 16 20

Microsoft PowerPoint - BCX2006 第三週 蛋白質定量

Microsoft PowerPoint - BCX2006 第三週蛋白質定量