雙向電泳D技術(shù)相關(guān)學(xué)習(xí)教案

1、雙向電泳雙向電泳D技術(shù)技術(shù)(jsh)相關(guān)相關(guān)第一頁，共22頁。Proteomics Experimental Workflow第1頁/共22頁第二頁，共22頁。Isoelectric focusing - IPG rehydrationIPGs are supplied as dry strips - on a flexible plastic supportThey must be rehydrated to at least their original volume (0.5 mm thick x 3.3 mm wide)Rehydration can be done in a focu

2、sing tray or a disposable rehydration tray.Focusing trayRehydration tray第2頁/共22頁第三頁，共22頁。First dimension what is the isoelectric point (pI)?until they reach the point in the pH gradient where their net charge is neutral. +-+-pH pIpH = pI第3頁/共22頁第四頁，共22頁。Proteins move in a pH gradient until they reac

3、h their isoelectric point (pI)Focusing with voltage91034567891034567893663543108784759476534910510869333344445555666667778889999101010Cathode-Anode+Anode+Cathode-pHpHFirst dimension what is isoelectric focusing (IEF)?第4頁/共22頁第五頁，共22頁。第5頁/共22頁第六頁，共22頁。第6頁/共22頁第七頁，共22頁。IPG stripsIPG strips are availab

4、le in a variety of pH gradients and lengthsReadyStrip IPG strips7 cm11 cm17 cm 18 cm24 cmLengthsBroad rangepH 3-10pH 3-10 nonlinear (NL)Narrow RangepH 3-6pH 4-7pH 5-8pH 7-10Micro RangepH 3.9-5.1pH 4.7-5.9pH 5.5-6.7pH 6.3-8.3第7頁/共22頁第八頁，共22頁。pH 3-10pH 3-10NL第8頁/共22頁第九頁，共22頁。IPG Strip Selection pH 3 -

5、 10 pH 7 - 10 pH 5 - 8 pH 3 - 6 pH 3-10pH 3-6pH 5-8pH 7-10E. coli Lysate 第9頁/共22頁第十頁，共22頁。IPG Strip ResolutionNarrowMicroBroad3-105-84.7-5.9第10頁/共22頁第十一頁，共22頁。第11頁/共22頁第十二頁，共22頁。ComponentPurposeTris HClBuffering AgentSDSDetergent: Disrupts hydrophobic interactions and gives the protein a net negativ

6、e charge GlycerolGive the sample weightME or DTTReducing AgentWhy do you use SDS in 1D SDS-PAGE buffer and not in rehydration/sample buffer?Why do you use glycerol in 1D SDS-PAGE buffer? Why do you use DTT in both 1D SDS-PAGE buffer and rehydration sample buffer? 第12頁/共22頁第十三頁，共22頁。 這些相互作用都包括什么呢？ co

7、valent interactions - disulfide bridges non-covalent interactions - ionic bonds, hydrogen bondshydrophobic interactions第13頁/共22頁第十四頁，共22頁。第14頁/共22頁第十五頁，共22頁。第15頁/共22頁第十六頁，共22頁。第16頁/共22頁第十七頁，共22頁。ComponentPurposeUreaChaotropic agent: Disrupts hydrogen bonds and prevents aggregation and formation of 2

8、 structures, helps solublizationDTTReducing agents: Disrupts disulphide bonds so proteins remain as single subunitsCHAPSDetergent: Disrupts hydrophobic interactions Carrier AmpholytesHelp counteract insufficient salt in a sampleBromophenol BlueAllows monitoring of the run第17頁/共22頁第十八頁，共22頁。Isoelectr

9、ic focusing rehydration & sample loadingPassive Rehydration: no voltage, sample is in rehydration bufferActive Rehydration: low voltage, sample is in rehydration bufferCup Loading: rehydrate with buffer, add sample to cup with low voltage第18頁/共22頁第十九頁，共22頁。TechniqueAdvantages DisadvantagesPassiv

10、e RehydrationSample application is simpleAvoids the problem of sample precipitationShorter focusing times can be used because the proteins are in the strip before focusing stepLarge amounts of proteins can be loadedThis allows the advantage of rehydrating in a separate tray while using the cell to f

11、ocus another batch of stripsLarge proteins may not enter the stripActive RehydrationSample application is simple Proteins enter gel by absorption and electrical pullAvoids the problem of sample precipitationShorter focusing times can be used because the proteins are in the strip before focusing step

12、Large amounts of proteins can be loadedsmall proteins with a higher mobility have a higher risk of being lost from the stripCup LoadingGood for samples that contain high levels of DNA, RNA or other large moleculesFor serum samples that have not been treated to remove albuminWhen running basic IPG strips (e.g. pH 7-10)For samples that contain high concentrations of glycoproteinsSample precipitation can occur More complicated, cup must be sealed to the surface of the pre-rehydrated empty stripIPG strip must be rehydrated prior to