双向电泳D技术相关学习教案

1、双向电泳双向电泳D技术技术(jsh)相关相关第一页，共22页。Proteomics Experimental Workflow第1页/共22页第二页，共22页。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页/共22页第三页，共22页。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页/共22页第四页，共22页。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页/共22页第五页，共22页。第5页/共22页第六页，共22页。第6页/共22页第七页，共22页。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页/共22页第八页，共22页。pH 3-10pH 3-10NL第8页/共22页第九页，共22页。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页/共22页第十页，共22页。IPG Strip ResolutionNarrowMicroBroad3-105-84.7-5.9第10页/共22页第十一页，共22页。第11页/共22页第十二页，共22页。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页/共22页第十三页，共22页。 这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页/共22页第十四页，共22页。第14页/共22页第十五页，共22页。第15页/共22页第十六页，共22页。第16页/共22页第十七页，共22页。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页/共22页第十八页，共22页。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页/共22页第十九页，共22页。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