電泳

1、蛋白質(zhì)電泳,－在蛋白質(zhì)組學(xué)中的應(yīng)用羅元明中科院微生物所,原 理,蛋白質(zhì)分子在溶液中由于其末端氨基、末端羧基及側(cè)鏈的游離基團而成為帶電顆粒，并可在電場內(nèi)移動，其移動方向取決于蛋白質(zhì)分子所帶靜電荷。不同蛋白質(zhì)分子根據(jù)其氨基酸組成及所在溶液的pH值，攜帶的靜電荷不盡相同，致使它們在電場中的遷移率各異，從而達到分理的目的。,電泳原理示意圖,在蛋白質(zhì)組學(xué)中對電泳的分類,一維電泳(one dimensional gel electropho

2、resis, 1DE)，現(xiàn)在普遍采用垂直板聚丙烯酰胺凝膠電泳（PAGE）二維電泳(two-dimensional gel electrophoresis, 2DE)，又稱雙向電泳,一維電泳,現(xiàn)在普遍采用聚丙烯酰胺凝膠電泳（PAGE），包括非變性電泳(native PAGE)和SDS-PAGE兩種，前者主要是在分離蛋白復(fù)合物時經(jīng)常用到。后者是在凝膠與緩沖系統(tǒng)中加入陰離子表面活性劑十二烷基硫酸鈉（SDS），蛋白質(zhì)分子被大量SDS陰離子包

3、裹，消除了它們間原來攜帶的電荷差別，因而其遷移率僅反映蛋白質(zhì)分子大小，故廣泛用于蛋白質(zhì)分子量的測定。,凝膠濃度和蛋白質(zhì)分離范圍,緩沖液的選擇,通常在SDS-PAGE均選擇Tris-glycine作為電泳的緩沖系統(tǒng)。但在大部分緩沖系統(tǒng)中，SDS微團(micelle)會干擾小分子蛋白質(zhì)的分離，而Tris-tricine系統(tǒng)則可使小蛋白質(zhì)－SDS復(fù)合物與微團分離，去除干擾。此外，也證明該系統(tǒng)對脂多糖和脂寡糖混合物的分離有效。,12%膠常用的低

4、分子量標(biāo)準(zhǔn)蛋白,一維凝膠染色,現(xiàn)在用于凝膠中蛋白染色的方法包括氨基黑10、考馬斯亮藍R250、考馬斯亮藍R350、銀染、銅染及橙染(sypro orange)。最常用的為考馬斯亮藍R250、考馬斯亮藍R350染色，為了提高靈明度采用銀染。,一維電泳的應(yīng)用,初步測定蛋白質(zhì)的分子量初步分離蛋白質(zhì)復(fù)合物，并進一步用于免疫組化分析對重組表達蛋白的初步鑒定將一維電泳和生物質(zhì)譜相結(jié)合，達到對較簡單蛋白復(fù)合物的分離和鑒定,一維電泳在蛋白質(zhì)組研究

5、中的應(yīng)用舉例,我們將一維電泳和LC-Ms/MS結(jié)合，成功進行如下研究： (1)血漿蛋白質(zhì)組研究 (2)SARS病毒蛋白的分析鑒定,成功鑒定了SARS病毒的S蛋白、N蛋白和E蛋白，有力推動了我國第一個SARS疫苗的研究和申報工作。,雙向電泳(2DE),原理：2DE就是第一向采用等電聚焦分離，第二向為SDS-PAGE分離。由于2DE是依據(jù)蛋白質(zhì)的兩種不同性質(zhì)，即等電點和分子量進行分析，因此分辨率遠高于其他任何一種單一的電泳方法，

6、是目前分析混合蛋白質(zhì)樣品最有效的手段。（注意：雙向電泳中的“雙向”指的是按照蛋白質(zhì)的兩個性質(zhì)即“等電點和分子量”進行分離的原理。),2DE儀器系統(tǒng),,,,,恒溫水浴,垂直板電泳儀（第二向）,電源,等電聚焦儀（第一向）,2DE在蛋白質(zhì)組學(xué)方案中所處的位置,,Functional analysis,,,,,,,,,,State 1,State 2,2DE,PMF,MS/MS,LC-MS/MS,Database search,,Differe

7、ntial analysis,?,,雙向電泳的操作程序(以進行差別表達蛋白組分析為例),1. 樣品制備2. IPG strips重泡脹及上樣(rehydration and liading)3. 等電聚焦4. 膠條平衡，包括還原和烷基化5. 第一向膠條轉(zhuǎn)移到第二向6. SDS-PAGE7. 染色8. 圖像分析,目前，普遍采用預(yù)制的固定pH梯度膠條(immobilized pH gradient strips, IPG s

8、trips)。商品化的IPG strips可以從Amersham Pharmacia公司或BioRad公司購買。,IPG膠條制備(casting of IPG strips),IPG slab gels with linear gradients pH 4-7, 4-9, 6-10 and 4-12 are cast according to Görg et al. (1986) with the recipes of Rig

9、hetti (1990) and Görg et al. (1998). Two starter solutions (an acidic one and a basic one) are prepared as described in Table 1. For better polymerization, the acidic and basic solutions are adjusted to pH 7 with s

10、odium hydroxide and acetic acid, respectively.,,Procedure,To assemble the polymerisation cassette wet the plain glass plate (size 260 x 200 mm2) with a few drops of water. Place the Gelbond PAGfilm, hydrophilic side upwa

11、rds, on the wetted surface of the plain glass plate. The GelBond PAGfilm should overlap the upper edge of the glass plate for 1-2 mm to facilitate filling of the cassette. Expel ecxess water with a roller. Place the glas

12、s plate which bears the U-frame (0.5 mm thick) on top of the GelBond PAGfilm and clamp the cassette together. Put it in the refrigerator for 30 min.,Assembly of the gel casting cassette,(Left): Assembly of the polymerisa

13、tion cassette for IPG and SDS gel casting on plastic backing (Glass plates, GelBond PAGfilm, U-frame 0.5 mm thick)(Right): Application of the GelBondPAGfilm onto the glass plate,IPG gel casting,(1) Gradient mixer and co

14、nnecting valve; (2) outlet tubing valve; (3) gel casting cassette,The acidic, dense solution is pipetted into the mixing chamber and the basic, light solution into the reservoir of the gradient mixer, an extra portion of

15、 the dense solution is prepared and pipetted into the mold prior to pouring the gradient.,After pouring the gradient into the precooled mold (refrigerator), the mold is kept at room temperature for 15 min to allow adequa

16、te levelling of the density gradient prior to polymerization for one hour at 50°C. After polymerization, the mold is kept at room temperature for at least 15 min. Then the IPG gel is removed from the mold and extens

17、ively washed with deionized water, impregnated with 2% glycerol, and dried at room temperature in a dust-free cabinet and, if not used immediately, covered with a plastic film for storage at -20°C. The dried gels ca

18、n be stored frozen for at least one year.,Note: In order to ensure the reproducibility of the IPG gradient, the volume of the cassette should be constant. Therefore, it is recommended to check the volume of the cassette

19、from time to time since it diminishes on ageing of the U-frame.,Note: When one of the chambers is emptying faster than the other, the resulting pH gradient will not be linear. Check if there is an air bubble in the conne

20、cting line or whether the speed of the magnetic stirrer is not appropriate!,1. 樣品制備,樣品裂解液(lysis solution): 8M urea, 4% CHAPS, 40 mM Tris base, 65 mMDTE 制備后的裂解液分裝凍存－20℃?zhèn)溆谩?如果必要，urea的濃度可增加到9或9.8M。 Triton X-100, NP

21、-40及其他的非離子型去污劑或Zwitterionic detergents可取代CHAPS,對于脂蛋白，膜蛋白的分析，可以用如下的蛋白質(zhì)裂解液：7M urea, 2M thiourea, 4% CHAPS, 40 mM Tris base, 65 mMDTE,Presulubilization of protein in (boiling) SDS buffer, followed by dilution with urea lys

22、is buffer.Initially solubilized in 0.5-1% SDS,followed by dilution with at least an eight excess of 2-4% (w/v) NP40, Triton X-100, or CHAPS to reduce the final concentration to <0.25%. This dilution displaces the SD

23、S from the proteins and replaces it with a nonionic or zwitterionic detergent.,制備2DE樣品時常見的污染物,鹽、小離子性分子(small ionic molecules)、離子型去污劑(ionic detergent)、核酸、多糖、脂類以及酚類化合物等，這些物質(zhì)會直接影響2DE的效果。,脂的去除,Lipids may interact with membra

24、ne proteins and consume detergents.High speed centrifugation of lipid-rich materialExtraction of the biological material with organic solvent (e.g.,ethanol or acetone)However, loss of proteins because certain proteins

25、 are soluble in the organic solvent or because the precipitated proteins do not always resolubilize,核酸的去除,Nucleic acids can interact with carrier ampholytes and proteins and give rise to 2D patterns containing horizontal

26、 streaks and increase the viscosity of the solution and may clog the pores of the gels.TCA/aceton (typically, 20%TCA in pure acetone) precipitation;Protease-free RNase and DNaseUltracentrifugation and addition of a ba

27、sic polyamine such as spermine;sonication,制備樣品的原則,樣品制備步驟越簡單越好，避免目的蛋白的損失或丟失。裂解細胞或組織要防止蛋白降解。裂解細胞應(yīng)在低溫下進行(冰水浴或4℃)，最好用加有蛋白酶抑制劑的裂解液直接裂解。樣品裂解液應(yīng)新鮮配制，或分裝凍存，而且要采用高純度的去離子尿素,蛋白樣品應(yīng)在等電聚焦前新鮮配制，或-80℃分裝凍存，絕對避免將樣品反復(fù)凍融。通過超離心去除所有顆粒性物質(zhì)，

28、因為顆粒性物質(zhì)或脂會阻塞凝膠孔路。,為了防止蛋白質(zhì)被修飾，加入尿素后，不要加熱蛋白樣品。當(dāng)樣品中含有尿素時，加熱絕對不要超過37℃.升高溫度會使尿素水解成異氰酸鹽(isocyanate),使蛋白發(fā)生氨甲?；?carbamylation)修飾。最好用強烈變性劑直接裂解樣品，這樣可以使蛋白水解酶立即變性失活。,細胞裂解方法,比較溫和的裂解方法包括： 滲透裂解(Osmotic lysis)，此法特別適合分離亞細胞組分，如血細胞、組織培

29、養(yǎng)細胞的裂解； 凍融裂解法(Freeze-thraw lysis)，快速將細胞凍于液氮中，然后融解，重復(fù)進行，如細菌、組織培養(yǎng)細胞的裂解；,去污劑裂解法(detergent lysis)，去污劑融解細胞膜，從而裂解細胞，釋放其中成分，如組織培養(yǎng)細胞的裂解； 酶裂解(enzymatic lysis)，具有細胞壁的細胞通過酶除去細胞壁。細胞用溶菌酶(lysozyme)，植物細胞用纖維素酶(cellulase), 果膠酶(pect

30、inase),酵母用溶細胞酶(lyticase)。,強烈的裂解方法包括：超聲波破碎，但要注意減少熱能和泡沫的產(chǎn)生，以防止蛋白質(zhì)變性或被剪切。手工研磨，?？梢约尤胧⑸??；虿捎醚欣?mortar)，或勻漿器(pestle)在液氮中研磨，如固體組織或微有機體的研磨。,蛋白酶抑制劑,苯甲基磺酰氟(PMSF, 1mM)，PMSF為酶的不可逆抑制劑，用于serine proteases及cysteine proteases的抑制。1mM

31、EDTA或1mMEGTAPeptide protease inhibitorsAEBSF (4mM)苯脒(Benzamidine) (1-3mM),其中，PMSF,EDTA及肽蛋白酶抑制劑, e.g., leupeptin(亮抑酶肽), pepstatin(胃蛋白酶抑制劑), aprotinin(抑制絲氨酸蛋白酶的堿性多肽), bestatin(亮氨酸類似物）是最常用的幾種蛋白酶抑制劑。,常用的蛋白質(zhì)沉淀方法,硫酸銨三氯乙酸(T

32、CA)丙酮，放于-20℃時效果會更好TCA＋丙酮,2. IPG 膠條重泡脹(rehydration)和上樣(loading),因為采用預(yù)制的IPG干膠條，需要用重泡脹液(rehydration solution)使其重性水化膨脹成凝膠，使樣品能夠充分進入凝膠內(nèi)，完成上樣。 可用重泡脹液在室溫浸泡12h后上樣，或在低電壓(30v)下室溫12h,使IPG干膠條邊水化膨脹邊上樣。,Rehydration solution,8 M urea

33、, 4% CHAPS, 100 mM DTT, 0.5-2% IPG buffer/pharmalyte,3. 第一向 等電聚焦(isoelectric focusing, IEF),,IPG Phor等電聚焦儀,,等電聚焦儀,典型的IEF條件舉例,采用安瑪西亞公司IPG Phor等電聚焦儀，pH 3-10的非線性膠條，蛋白上樣量250μg，體積350μl。電流為50μA／strip，所用電泳條件為30V,12h;100v,1h; 5

34、00v, 1h;1000v,1h; 8000v,1h(gradient); 8000v,10h，共用時26h，IEF結(jié)束后vhs為80000左右。,4. IEF后膠條的平衡,SDS平衡緩沖液為：50mM Tris-Cl pH8.8, 6M Urea, 30% glycerol, 2% SDS, trace bromophenol 還原：100mg DTT/10 ml SDS 平衡buffer，15min; 烷基化：250mg

35、 iodoacetamide/10ml SDS平衡buffer, 15min.,5. 膠條向第二向轉(zhuǎn)移,將IEF后的膠條轉(zhuǎn)移到垂直板聚丙烯酰胺上，用1%的瓊脂糖封閉，從而使膠條上的蛋白轉(zhuǎn)移到第二向分離膠中，根據(jù)分子量大小將pI接近的蛋白進行進一步分離。,注意：進行第二向SDS-PAGE時，要求恒溫，但溫度不要低于15℃，否則會引起凝膠收縮，影響蛋白從膠條向第二向凝膠的轉(zhuǎn)移。,6. SDS-PAGE,7. 2D膠的染色,考馬斯亮藍染色，常

36、用考馬斯亮藍R250,R350(sensitivity: 1ug/spot)負(fù)染(negative staining), 如鋅染 (ST: 50 ng)銀染，為了有利于對2D膠上的蛋白進行質(zhì)譜鑒定，最好不要使用戊二醛，因為戊二醛會和蛋白質(zhì)交聯(lián)，降低肽提取效率(0.1 ng)。熒光染色，為了提高靈敏度，常采用熒光染料染色,e.g., SYPRO Ruby, ST:1-2 ng, single step, available for

37、automation放射性同位素染色(125I,32P, 14C or 35S),標(biāo)記的蛋白質(zhì)直接進行放射自顯影,Comparison of fluorescent labelled 2-D gel with silver staining,8. 圖像分析,在用雙向電泳進行差別表達蛋白質(zhì)組分析時，需要利用軟件對產(chǎn)生的2D膠進行差別分析，以尋找差別表達蛋白。常用的軟件如安瑪西亞公司的Image Master 2D Elite或Image

38、 Master 2D Planium分析軟件。,圖像分析一般程序,Image visualization and manipulationSpot detection and quantificationSpot matchingEditing matchesMolecular weight and pI calibrationSynthetic and average gelsAnalyzing the dataAnno

39、tating the gels,Image analysis with ImageMaster Platinum,Image analysis with ImageMaster Platinum,,Spots report,差別分析(Differential analysis),表達增高的點（Up-regulated spots）表達降低的點（Down-regulated spots）只在樣品或?qū)φ掌分谐霈F(xiàn)的特異性點,差別分析示意圖

40、,Sample 1 a,Sample 1 b,Sample 1 c,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,表達上調(diào),表達下調(diào)的點(right),,,,2DE常見問題及解決辦法(Troubleshooting),Too much salt in the sample (disturbs IEF)Hint:Include an acetone precipitation to rem

41、ove salts and other contaminants,Charged impurities in the sampleHint:Include an acetone precipitation to remove salts and other contaminants,Impurities in the sample or rehydration solutionHint:Include an acetone pr

42、ecipitation to remove salts and other contaminantsPrepare new rehydration solution with pure reagents,underfocused (Focusing time not long enough)Hint:Prolong the focusing time of the IEF,Gel surface during polymeriza

43、tion not overlaid with destilled water (or too low amount of water used)Hint:Apply at least 1 ml to overlay the gel surface,No uniform gel polymerization (e.g. impurities at gel cassettes, air bubbles in the polymerize

44、d gel)Hint:Clean gel cassettes with ethanolCast the slab gel slowly (approx. 1 min)Overlay the gel carefully with destilled water,Not all proteins (especially high molecular mass proteins) saturated with SDSHint:Us

45、e 0.15 % instead of 0.1 % (w/ v) SDS in the 1 x SDS buffer for SDS-PAGE,High sample load (protein disturbs separation of other spots or may not be fully saturated with SDS)Hint:Lower the protein amount,Impurities on or

46、 within the 2-D gel still present during silver stainingHint:Clean the gel cassettes prior casting with ethanolIncubate the 2-D gels long enough (and with at least 100 ml/ gel) in fixing and washing solution prior sta

47、ining,進行2DE分析幾點建議,樣品制備是關(guān)鍵，務(wù)必使蛋白樣品充分裂解，除去顆粒性物質(zhì)，如核酸、糖，鹽及去污劑等的影響要注意蛋白從第一向到第二向的轉(zhuǎn)移，可用分離膠直接封閉IPG膠條為了提高銀染的效果，玻璃板和染色盤一定要清洗干凈如要進行差別蛋白組分析，銀染的方法要注意和質(zhì)譜分析相匹配,提高2DE分辨率的策略,首先采用寬pH范圍的膠條，確定蛋白的分布范圍，通常采用pH3-10的膠條如果pH線性分布的膠條分離效果不好，可采用非線

48、性膠條加大蛋白上樣量，提高局部蛋白的分辨率采用窄pH范圍的膠條，提高某pH范圍蛋白的分辨率第二向采用梯度膠進行分離去除高豐度蛋白，進行蛋白的fractionation處理,富集低豐度蛋白,采用窄范圍pH膠條對pH4-7的2D膠進行局部分辨率提高的分析舉例,pH4-7膠條分析的2D膠圖譜,,pH4-5膠條分析的2D膠圖譜,pH4-5膠條分析的2D膠圖譜,pH5.5-6.7膠條分析的2D膠圖譜,如上所示：通過分別用pH4-5,pH

49、4-6,pH5.5-6.7的窄pH范圍膠條，較單獨用pH4-7的膠條，可大大提高局部區(qū)域的分辨率，使檢測到的點數(shù)大大增加。,Pre-及subfractionation方法,Isolation of cell compartments and /or organelles, e.g., by sucrose gradient centrifugation;Selective precipitation of certain protei

50、n classes (e.g., TCA/acetone);Sequential extraction with increasingly powerful solubilizing buffers,e.g., aqueous buffers, organic solvents such as ethanol or chloroform/methanol, and detergent-based extraction solution

51、Chromatographic or electrokinetic separation methods,膠內(nèi)差別電泳(difference in gel electrophoresis, DIGE),一種改進的進行差別分析的2DE方法,DIGE分析特點,將樣品和對照品用熒光染料進行標(biāo)記，在同一塊膠內(nèi)，對樣品和對照品進行雙向電泳分析，為一種改進的2DE方法。,DIGE分析流程圖,DIGE的優(yōu)點,在DIGE時，由于樣品和對照在同一膠內(nèi)分

52、離，使用相同的內(nèi)標(biāo)，避免了使用不同凝膠時在操作上的偶然性和不平行性，可以準(zhǔn)確檢測出兩個不同樣品蛋白表達上的差別，消除膠與膠之間的差異，保證統(tǒng)計上的可靠性及操作上的重復(fù)性。熒光標(biāo)記較其他染色方法靈敏度更高。與常規(guī)的雙向電泳技術(shù)比較，DIGE更加簡單，勞動強度大大降低，效率大大提高。,雙向電泳的應(yīng)用,分離復(fù)雜的蛋白質(zhì)組：包括系統(tǒng)分離(systematic separation)，鑒定(identification)及定量(quantific

53、ation);預(yù)測蛋白質(zhì)的翻譯后修飾；差別表達蛋白質(zhì)組分析，研究細胞分化、尋找疾病相關(guān)的生物標(biāo)記分子、進行疾病治療情況的監(jiān)測、藥物開發(fā)及癌癥研究等。,2DE的局限性,The results are difficult to reproduceIt has a limited dynamic range and is difficult to audomate.It is labor intensivealternative m