第三章蛋白質(zhì)5周五

上節(jié)內(nèi)容蛋白質(zhì)的結(jié)構(gòu)與幾種蛋白質(zhì)的結(jié)構(gòu)與轉(zhuǎn)運(yùn)蛋轉(zhuǎn)運(yùn)蛋收縮與免疫球蛋白質(zhì)兩性膠沉兩性膠沉變性與折紫外本章主要內(nèi)二．氨基重點(diǎn))四．蛋白質(zhì)的結(jié)構(gòu)（重點(diǎn)五．蛋白質(zhì)結(jié)構(gòu)與功能（重點(diǎn)六．蛋白質(zhì)的性質(zhì)（重點(diǎn)七．蛋白質(zhì)的分離純化與鑒定（重點(diǎn)七、Protein1.Salting鹽析（saltingout）原理：破壞蛋白質(zhì)疏水區(qū)表面的水化層導(dǎo)致蛋白質(zhì)沉淀。最先的是那些表面Differentproteinsprecipitatedatdifferentsaltconcentrations(飽和或半飽和濃度).Itisoneofthemostcommonlyusedforprotein鹽溶（Saltingin）稀鹽濃度范圍內(nèi)，增加蛋白質(zhì)SaltingAmmoniumsulfate,(NH4)2SO4,isthemostcommonlyusedreagentforsaltingoutThepHmaybeadjustedtoapproximatethepIofthedesiredprotein.Asignificantconcentration（濃縮）oflargetiesofproteinSalting分分級(jí)增加鹽濃度，控制pH,沉淀目標(biāo)蛋白（白色）Ionexchange原理：電荷差異，參看aminoAnion陰離子DEAE:Cation陽(yáng)離子exchangersCM:carboxymethylMatrixcelluloseandagarose-basedresins樹(shù)脂IonexchangeIsoelectric等電聚焦（IEF）原理：電荷不可用于蛋白質(zhì)等電點(diǎn)的測(cè)定，分辨率（pI<GelfiltrationMolecularsieve分子篩MolecularareseparatedaccordingtotheirsizeandshapeStationaryphase固定相gelbeadscontainingporesthatspanarelativelynarrowsizerangeSephadex（葡聚糖凝膠Bio-gelP（聚丙烯酰胺凝膠Gelfiltration七、ProteinSDSsodiumdodecylsulfate十二烷基磺酸鈉PAGEpolyacrylamidegelelectrophoresis非常有純度檢測(cè)；亞基組成分析；分子量測(cè)1.4gSDS/1g兩個(gè)氨基酸結(jié)合一個(gè) 結(jié)果所有肽鏈覆蓋上相同密度的負(fù)電荷可看成若存在鏈間二硫鍵，如何進(jìn)行分SDS裝置及電泳SDS測(cè)定蛋白質(zhì)的Two-dimensionalPermitstheresolutionofcomplexmixturesofproteinsAmoresensitive yticalmethodthaneitherelectrophoreticmethodalone.SeparatesproteinsofidenticalmolecularweightthatdifferinpI,orproteinswithsimilarpIvaluesbutdifferentmolecularTwo-dimensional②①>60000應(yīng)用：最常用方法：(densitygradient常用的密度梯度：DensityGradient（密度梯度離心Dialysisand半透膜：玻璃紙、火棉紙、改性纖維素將蛋白質(zhì)與其他小分子物濃縮和脫粗分Ultrafiltration(超過(guò)濾Affinity（親和色譜Principle:Manyproteinscanbindspecificmolecules(ligand)tightly.Ligandiscovalentlyattachedtoaninertmatrix.Asignificantpurificationchromatography(免疫親和色譜)ligand=antibodyHis-tag（組氨 其他用途增加蛋白可溶性提高蛋白穩(wěn)定性漿狀動(dòng)(植)機(jī)械破漿狀動(dòng)(植)

低速離蛋白(除膜等雜蛋白蛋白蛋白

蛋蛋白層析電泳檢蛋白蛋白

純純稀透（除鹽

電泳檢

－80℃蛋白質(zhì)含量雙縮Folin－酚試劑法（Lowry法Bradford法（考馬斯亮 蛋白質(zhì)純度HP