實驗操作指導(dǎo)書：蛋白質(zhì)的聚丙烯酰胺凝膠電泳

1、PAGE PAGE 206實驗四 蛋白質(zhì)的聚丙烯酰胺凝膠電泳最廣泛使用的不連續(xù)緩沖系統(tǒng)最早是由Ornstein(1964) 和Davis(1964) 設(shè)計的, 樣品和濃縮膠中含 Tris-HCl(pH 6.8), 上下槽緩沖液含Tris-甘氨酸(pH 8.3), 分離膠中含Tris-HCl(pH 8.8)。系統(tǒng)中所有組分都含有0.1% 的 SDS(Laemmli, 1970)。樣品和濃縮膠中的氯離子形成移動界面的先導(dǎo)邊界而甘氨酸分子則組成尾隨邊界，在移動界面的兩邊界之間是一電導(dǎo)較低而電位滴度較陡的區(qū)域, 它推動樣品中的蛋白質(zhì)前移并在分離膠前沿積聚。此處pH值較高， 有利于甘氨酸的離子化，所形成

2、的甘氨酸離子穿過堆集的蛋白質(zhì)并緊隨氯離子之后，沿分離膠泳動。從移動界面中解脫后，SDS-蛋白質(zhì)復(fù)合物成一電位和pH值均勻的區(qū)帶泳動穿過分離膠，并被篩分而依各自的大小得到分離。SDS與蛋白質(zhì)結(jié)合后引起蛋白質(zhì)構(gòu)象的改變。SDS-蛋白質(zhì)復(fù)合物的流體力學(xué)和光學(xué)性質(zhì)表明，它們在水溶液中的形狀，近似于雪茄煙形狀的長橢園棒，不同蛋白質(zhì)的SDS復(fù)合物的短軸長度都一樣（約為18，即1.8nm），而長軸則隨蛋白質(zhì)分子量成正比地變化。這樣的SDS-蛋白質(zhì)復(fù)合物，在凝膠電泳中的遷移率，不再受蛋白質(zhì)原有電荷和形狀的影響，而只是橢園棒的長度也就是蛋白質(zhì)分子量的函數(shù)。由于SDS和巰基乙醇的作用，蛋白質(zhì)完全變性和解聚，解離成

3、亞基或單個肽鏈，因此測定的結(jié)果只是亞基或單條肽鏈的分子量。SDS聚丙烯酰胺凝膠的有效分離笵圍取決于用于灌膠的聚丙烯酰胺的濃度和交聯(lián)度。在沒有交聯(lián)劑的情況下聚合的丙烯酰胺形成毫無價值的粘稠溶液，而經(jīng)雙丙烯酰胺交聯(lián)后凝膠的剛性和抗張強度都有所增加，并形成SDS蛋白質(zhì)復(fù)合物必須通過的小孔。這些小孔的孔徑隨 “雙丙烯酰胺丙烯酰胺” 比率的增加而變小，比率接近 1：20 時孔徑達到最小值。SDS聚丙烯酰胺凝膠大多按“雙丙烯酰胺丙烯酰胺”為1：29 配制，試驗表明它能分離大小相差只有3% 的蛋白質(zhì)。凝膠的篩分特性取決于它的孔徑，而孔徑又是灌膠時所用丙烯酰胺和雙丙烯酰胺絕對濃度的函數(shù)。用515%的丙烯酰胺所

4、灌制凝膠的線性分離范圍如下表：SDS聚丙烯酰胺凝膠的有效分離范圍*丙烯酰胺濃度（%）線性分離范圍（kD）1512431016687.536945.057212*雙丙烯酰胺丙烯酰胺摩爾比為 1：29。1. SDS聚丙烯酰胺凝膠的配制 試劑丙烯酰胺和N, N-亞甲雙丙烯酰胺。以溫?zé)幔ɡ谌芙怆p丙烯酰胺）的去離子水配制含有29%（w/v）丙烯酰胺和1%（w/v）N, N-亞甲雙丙烯酰胺的貯存液，丙烯酰胺和雙丙烯酰胺在貯存過程中緩慢轉(zhuǎn)變?yōu)楸┧岷碗p丙烯酸，這一脫氨基反應(yīng)是光催化或堿催化的，故應(yīng)核實溶液的pH值不超過7.0。這一溶液置棕色瓶中貯存于室溫，每隔幾個月須重新配制。小心：丙烯酰胺和雙丙烯酰胺具

5、有很強的神經(jīng)毒性并容易吸附于皮膚。 十二烷基硫酸鈉（SDS）。SDS可用去離子水配成10%（w/v）貯存液保存于室溫。用于制備分離膠和積層膠的Tris緩沖液。TEMED(N,N,N,N-四甲基乙二胺)。TEMED通過催化過硫酸銨形成自由基而加速丙烯酰胺與雙丙烯酰胺的聚合。過硫酸銨。 過硫酸銨提供驅(qū)動丙烯酰胺和雙丙烯酰胺聚合所必需的自由基。須新鮮配制。Tris-甘氨酸電泳緩沖液。 裝置： 使用不連續(xù)緩沖系統(tǒng)要求在垂直板凝膠上進行SDS聚丙烯酰胺電泳。2SDS聚丙烯酰胺凝膠的灌制 根據(jù)廠家說明書安裝玻璃板。 確定所需凝膠溶液體積，按下表給出的數(shù)值在一小燒杯中按所需丙烯酰胺濃度配制一定體積的分離膠溶

6、液。一旦加入TEMED，馬上開始聚合，故應(yīng)立即快速旋動混合物并進入下步操作。配制Tris-甘氨酸SDS聚丙烯酰胺凝膠電泳分離膠溶液溶液成分總體積 5ml總體積 10ml總體積 15ml總體積 20ml總體積 25ml總體積 30ml6%水2.6ml5.3ml7.9ml10.6ml13.2ml15.9ml30%丙烯酰胺1.0ml2.0ml3.0ml4.0ml5.0ml6.0ml1.5M Tris(pH 8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10% SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%過硫酸胺0.05ml0.1ml0.15ml

7、0.2ml0.25ml0.3mlTEMED0.004ml0.008ml0.012ml0.016ml0.02ml0.024ml8%水2.3ml4.6ml6.9ml9.3ml11.5ml13.9ml30%丙烯酰胺1.3ml2.7ml4.0ml5.3ml6.7ml8.0ml1.5M Tris(pH 8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10% SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%過硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.003ml0.006ml0.009ml0.012ml0.015m

8、l0.018ml10%水1.9ml4.0ml5.9ml7.9ml9.9ml11.9ml30%丙烯酰胺1.7ml3.3ml5.0ml6.7ml8.3ml10.0ml1.5M Tris(pH 8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10% SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%過硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.002ml0.004ml0.006ml0.008ml0.01ml0.012ml12%水1.6ml3.3ml4.9ml6.6ml8.2ml9.9ml30%丙烯酰胺2.0ml

9、4.0ml6.0ml8.0ml10.0ml12.0ml1.5M Tris(pH 8.8)1.3ml2.5ml3.8ml5.0ml6.3ml7.5ml10% SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%過硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.002ml0.004ml0.006ml0.008ml0.01ml0.012ml15%水1.1ml2.3ml3.4ml4.6ml5.7ml6.9ml30%丙烯酰胺2.5ml5.0ml7.5ml10.0ml12.5ml15.0ml1.5M Tris(pH 8.8)1.3ml2.5m

10、l3.8ml5.0ml6.3ml7.5ml10% SDS0.05ml0.1ml0.15ml0.2ml0.25ml0.3ml10%過硫酸胺0.05ml0.1ml0.15ml0.2ml0.25ml0.3mlTEMED0.002ml0.004ml0.006ml0.008ml0.01ml0.012ml 迅速在兩玻璃板的間隙中灌注丙烯酰胺溶液,留出灌注濃縮膠所需空間(梳子的齒長再加0.5cm)。再在膠液面上小心注入一層水（約23mm高），以阻止氧氣進入凝膠溶液。 分離膠聚合完全后（約30分鐘），傾出覆蓋水層，再用濾紙吸凈殘留水。 制備濃縮膠：按下表給出的數(shù)據(jù)，在另一小燒杯中制備一定體積及一定濃度的丙烯酰

11、胺溶液，一旦加入TEMED，馬上開始聚合，故應(yīng)立即快速旋動混合物并進入下步操作。配制Tris-甘氨酸 SDS聚丙烯酰胺凝膠電泳5% 濃縮膠溶液溶液成分總體積 3ml總體積 4ml總體積 5ml總體積 6ml總體積 8ml水2.1ml2.7ml3.4ml4.1ml5.5ml30%丙烯酰胺0.5ml0.67ml0.83ml1.0ml1.3ml1M Tris(pH 6.8)0.38ml0.5ml0.63ml0.75ml1.0ml10% SDS0.03ml0.04ml0.05ml0.06ml0.08ml10%過硫酸胺0.03ml0.04ml0.05ml0.06ml0.08mlTEMED0.003ml0

12、.004ml0.005ml0.006ml0.008ml聚合的分離膠上直接灌注濃縮膠,立即在濃縮膠溶液中插入干凈的梳子。小心避免混入氣泡，再加入濃縮膠溶液以充滿梳子之間的空隙，將凝膠垂直放置于室溫下。在等待濃縮膠聚合時，可對樣品進行處理，在樣品中按1:1體積比加入樣品處理液，在100加熱3分鐘以使蛋白質(zhì)變性。樣品處理液配方：50mM Tris-HCl(pH 6.8)100mM DTT(or 5% 巰基乙醇)2% SDS0.1% 溴酚藍10%甘油 濃縮膠聚合完全后（30分鐘），小心移出梳子。把凝膠固定于電泳裝置上，上下槽各加入Tris-甘氨酸電極緩沖液。必須設(shè)法排出凝膠底部兩玻璃板之間的氣泡。Tr

13、is-甘氨酸電極緩沖液：25mM Tris250mM 甘氨酸 (pH 8.3)0.1% SDS 按予定順序加樣，加樣量通常為1025l（1.5mm厚的膠）。 將電泳裝置與電源相接，凝膠上所加電壓為8V/cm。當(dāng)染料前沿進入分離膠后，把電壓提高到15V/cm，繼續(xù)電泳直至溴酚藍到達分離膠底部上方約1cm，然后關(guān)閉電源。 從電泳裝置上卸下玻璃板，用刮勺撬開玻璃板。緊靠最左邊一孔（第一槽）凝膠下部切去一角以標(biāo)注凝膠的方位。3用考馬斯亮藍對SDS聚丙烯酰胺凝膠進行染色經(jīng)SDS聚丙烯酰胺凝膠電泳分離的蛋白質(zhì)樣品可用考馬斯亮藍R250染色。染色液：0.1% 考馬斯亮藍 R25040% 甲醇10% 冰醋酸染

14、色12小時或過夜。脫色液：10% 甲醇10% 冰醋酸脫色需310小時，其間更換多次脫色液至背景清楚。此方法檢測靈敏度為0.21.0g。脫色后，可將凝膠浸于水中，長期封裝在塑料袋內(nèi)而不降低染色強度。為永久性記錄，可對凝膠進行拍照，或?qū)⒛z干燥成膠片。4. 測量并計算分子量蛋白質(zhì)的分子量與它的電泳遷移有一定關(guān)系式，經(jīng)37種蛋白的測定得到以下的關(guān)系式： Mw K (10bm) (1) lgMw = lg K bm = K1bm (2) 其中Mw是蛋白質(zhì)分子量；K和K1為常數(shù) b為斜率，m 是電泳遷移率，實際使用的是相對遷移率mR。如果用幾種標(biāo)準(zhǔn)蛋白質(zhì)分子量的對數(shù)作縱坐標(biāo)，用各自的相對遷移率作橫坐標(biāo)，

15、即可畫出一條斜率為負的標(biāo)準(zhǔn)曲線。相對遷移率為： 其中，dpr、dBPB分別為樣品和BPB（溴酚蘭）以分離膠表面為起點遷移的距離。 欲求未知蛋白的分子量，只需求出它的相對遷移率： mR未= dpr未/ dBPB 然后，從標(biāo)準(zhǔn)曲線上就可求出此未知蛋白的分子量。取出脫色后的凝膠平放在兩塊透明投影膠片中間，趕盡氣泡，在復(fù)印機上復(fù)印。在復(fù)印的凝膠圖上用直尺分別量出各條蛋白帶遷移的距離dpr和dBPB（以蛋白帶的上沿或中心為準(zhǔn)），計算相對遷移率，根據(jù)方程式： lgMw = K1bmR 用各標(biāo)準(zhǔn)蛋白分子量的對數(shù)（縱坐標(biāo)）和相對遷移率mR（橫坐標(biāo)）畫出標(biāo)準(zhǔn)曲線，由標(biāo)準(zhǔn)曲線再求出其他各條待測和未知蛋白帶的分子量

16、，如有可能計算其誤差。 SDSI. BACKGROUND:Anionic surfactant compounds such as sodium dodecyl sulfate(SDS) are composed of a negatively charged ionic “head group” and a hydrophobic hydrocarbon “tail”. CH3CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 OSO3 Na+ The high solubility in water imparted by the ionic head

17、group and the high solubility in nonpolar solvents implied by the hydrocarbon tail result in a compromise in aqueous solution. Surfactant molecules form aggregates called micelles in aqueous solution. These aggregates satisfy the solubility characteristics of both the head and tail regions of the su

18、rfactant; ionic groups are exposed to water on the surface of the aggregate while the hydrophobic tails associate with each other within the interior of a roughly spherical aggregate of 60 to 100 molecules. Hydrophobic guest molecules can be taken into the interior of the micelle. This includes the

19、hydrophobic amino acids normally confined to the interior of a native protein. The association between hydrophobic amino acid residues and the micelle interior is strong enough to denature most proteins, turning them inside-out so that become effectively coated with anionic surfactant molecules.Redu

20、ctive cleavage of all S-S- bonds followed by treatment with the anionic surfactant sodium dodecyl sulfate(SDS) will disrupt the native tertiary structure of most proteins, causing them to adopt rodlike structures. It has been established that this binding occurs with a constant surfactant-totprotein

21、 weight ratio and with enough anionic surfactants to totally dominate the native charge of the protein. Therefore, the charge per unit protein weight is nearly constant and the electrophoretic mobility of SDS denatured proteins is a function of size alone. The technique of SDS-polyacrylamide gel ele

22、ctrophoresis is widely used to determine the molecular weight of unknown proteins by comparing their relative electrophoretic mobility to standard proteins of known molecular weight. A direct comparison of the mobilities of known and unknown proteins run under identical conditions (in the same cell

23、at the same time) is recommended. The mobilities of known proteins can be plotted as a function of log(molecular weight) and the mobilities of known proteins used to estimate molecular weights by extrapolation.A necessary first step in the protein sample preparation for SDS electrophoresis is treatm

24、ent with an excess of 2-mercaptoethanol, which reduces all disulfide(-S-S-) bonds in the protein. This permits total disruption of the protein native structure, which is usually stabilized by disulfide linkages. Some proteins(e.g., chymotrypsin) contain polypeptides linked only by disulfide bonds. R

25、eduction will yield two or more polypeptide fragments which will migrate independently. In the same way oligomeric proteins(e.g., hemoglobin) will dissociate into monomeric subunits when solubilized by SDS. For this reason, hemoglobin migrates as a monomer of molecular weight 16,000 rather than a te

26、tramer of 64,500 D. The subunit dissociation caused by SDS is one reason why oligomeric proteins must be characterized by both SDS and disc electrophoresis. The pore size of the gel network is the critical factor in SDSseparations. The average pore size can be decreased by either increasing the tota

27、l concentration of monomer(both acrylamide and BIS) or by increasing the proportion of cross-linker(BIS) to the total monomer in the gel. Table 1 gives the approximate molecular weight ranges that can be conveniently estimated with gels of various total monomer concentrations, expressed as percent-g

28、el.%gel = (g Acrylamide + g BIS)/ 100ml x 100Table 1. MOLECULAR WEIGHT RANGES FOR SDSTotal Acrylamide(% gel)15105M.W. Range(Kilodaltons)3-5010-7025-200This methods separates proteins based primarily on their molecular weights (Laemmli, 1970). Among the varied uses of this technique are:Analysis of p

29、rotein purity;Determination of protein molecular weight;Verification of protein concentration;Detection of proteolysis;Identification of immunoprecipitated proteins;First stage of immunoblotting;Detection of protein modification ;Separation and concentration of protein antigens for antibody producti

30、on;Separation of radioactively labeled proteins.Sensitivity of staining:Coomassie Blue: 0.11 g per band (Smith, 1984);Silver Staining: 210 ng per band (Giulian et al., 1983).Time Required:Individual Steps:Pouring Separating Gel:60 minutesPouring Stacking Gel:30 minutesLoading Samples:15 minutesElect

31、rophoresis:45 minutesStaining:Coomassie Staining: 30 minutes (major bands), 3 hours for major bands to destain. Complete destaining may require 24-48 hours.Silver Staining: 6 hours.II. BUFFER SYSTEM:(A): Lower buffer: 18.17g Tris,0.4g SDS, pH 8.8 (HCl),Added H2O to 100ml (1.5M Tris-HCl buffer). (B):

32、 Upper buffer: 6.06g Tris,0.4g SDS, pH 6.8 (HCl),Added H2O to 100ml (0.5M Tris-HCl buffer).(C): 30% Acrylamide: 30g Acrylamide,0.8g Bisacrylamide,Added H2O to 100ml.(D): 10%(w/v) Ammonium persulfate (fresh):0.1g ammonium persulfate, Added H2O to 1ml.(F): Electrophoresis Buffer:3g Tris,14.4g glycine,

33、1g SDS,Added H2O to 1 liter.*H2O: Distilled water.*Amounts of Working Solutions to UseVolumes necessary for pouring gels of different thickness (for two 6x8cm gels)Gel ThicknessSeparatingStacking0.5mm5.6ml1.4ml0.75mm8.4ml2.1ml1.0mm11.2ml2.8ml1.5mm16.8ml4.2mlAlways prepare with a moderate excess of g

34、el solution.Calculation for x% Separating Gel:Solution (C) x/3 mlSolution (A)2.5 mlH2O(7.5-x/3) ml10% Ammonium Persulfate50lTEMED5l*Total Volume 10 ml.*Pouring the Separating GelExample of Separating Gel Preparation:12.5% Running gel (Separating gel):A4.5mlC7.5mlH2O5.9mlTEMED0.02ml D(fresh)0.07mlTot

35、al18mlTEMED or D was added last.Assemble gel sandwich according to the manufacturers instructions, or according to the usage of alternative systems. For Mini-Gel, be sure that the bottom of both gel plates and spacers are perfectly flush against a flat surface before tightening clamp assembly. A sli

36、ght misalignment will result in a leak.Combine solutions C and B and water in a small Erlenmeyer flask or test tube.Add ammonium persulfate and TEMED, and mix by swirling or inverting container gently (excessive aeration will interfere with polymerization). Work rapidly at this point because polymer

37、ization will be under way.Carefully introduce solution into gel sandwich using a pipet. Pipet solution so that it descends along a spacer. This minimizes the possibility of air bubbles becoming trapped with the gel.When the appropriate amount of separating gel solution has been added (in the case of

38、 the Mini-Gel, about 1.5cm from top of front plate or 0.5cm below level where teeth of comb will reach, Fig 1), gently layer about 1cm of water on top of the separating gel solution. This keeps the gel surface flat.Allow gel to polymerize (30-60 minutes). When the gel has polymerized, a distinct int

39、erface will appear between the separating gel and the water, and the gel mold can be tilted to verify polymerization.Fig 1. Separating gel prior to polymerization. *Pouring the Stacking GelExample of Stacking Gel PreparationStacking gel (3%):B2.0mlC0.8mlH2O5.2mlTEMED0.01ml D(fresh)0.04mlTotal8.05mlT

40、EMED or D was added last.Pour off water covering the separating gel. The small droplets remaining will not disturb the stacking gel.Combine Solution C and B and water in a small Erlenmeyer flask or a test tube.Add ammonium persulfate and TEMED and mix by gently swirling or inverting the container.Pi

41、pet stacking gel solution onto separating gel until solution reaches top of front plate.Carefully insert comb into gel sandwich until bottom of teeth reach top of front plate (Fig 2). Be sure no bubbles are trapped on ends of teeth. Tilting the comb at a slight angle is helpful for insertion without

42、 trapping air bubbles. Allow stacking gel to polymerize (about 30 minutes).After stacking gel has polymerized, remove comb carefully (making sure not to tear the well ears).Place gel into electrophoresis chamber. Add electrophoresis buffer to inner and outer reservoir, making sure that both top and

43、bottom of gel are immersed in buffer.Fig 2. Inserting the sample-well comb into the stacking gel.III.PROTEIN SAMPLE PREPARATION:1. Sample buffer: 1g SDS, 5ml Glycerol,0.5mg Bromophenol blue,2.5ml Mercaptoethanol,10ml Upper buffer,Added H2O to 50ml.Protein sample is diluted with an equal volume of sa

44、mple buffer. The mixture is heated in a boiling-water bath for 5 minutes. The amount of protein to be applied depends on the sample but applications in the range 15g of protein per protein band usually give sharp, easily viewed bands. Sample volumes of 10100l are most common. *Capacity per Well (Min

45、i-Gel System)Gel Thickness1 Well5 Wells10 Wells15 Wells0.5mm0.7ml45l16l9l0.75mm1.0ml68l24l14l1.0mm1.4ml90l32l18l1.5mm2.1ml135l48l27l*Introduce sample solution into well using a Hamilton syringe (Fig 3). Layer protein solution on bottom of well and raise syringe tip as dye level rises. Be careful to

46、avoid introducing air bubbles as this may allow some of sample to be carried to adjacent well.*Rinse syringe thoroughly with electrode buffer or water before loading different samples. Include molecular weight standards in one or both outside wells.Fig 3. Introducing protein solution into sample wel

47、l.2. Molecular Weight Standards:A slab gel is especially useful for molecular weight determinations since the sample and molecular weight standard proteins can be run under identical conditions on a single gel. There are a number of commercially available SDSmolecular weight standards which give a g

48、ood spread of molecular weight lines in a gel.*Molecular Weight Standards Mid-Range Protein:ProteinMolecular Weight(kD)Phosphorylase b(Rabbit Muscle)97.4BSA(Bovine Serum Albumin)66Ovalbumin(Chicken Egg)43Carbonic anhydrase(Bovine Erythrocytes)31Lysozyme(Chicken Egg White)14.4IV. SLAB ELECTROPHORESIS CONDITIONS:Running a Gel *StepsAttach ele