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1、2D Electrophoresis: 1st Dimension,Proteomics Experimental Workflow,Isoelectric focusing - IPG rehydration,IPGs 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 focusing tray or
2、 a disposable rehydration tray.,Focusing tray,Rehydration tray,First dimension what is the isoelectric point (pI)?,Proteins are amphoteric The charges are determined by amino acid composition pH of the environment Isoelectric point IEF is electrophoresis of proteins in a pH gradient. Charged protein
3、s move in an electric field until they reach the point in the pH gradient where their net charge is neutral.,Proteins move in a pH gradient until they reach their isoelectric point (pI),Focusing with voltage,9,3,6,6,3,5,4,3,10,8,7,8,4,7,5,9,4,7,6,5,3,4,9,10,5,10,8,6,9,3,3,3,3,4,4,4,4,5,5,5,5,6,6,6,6
4、,6,7,7,7,8,8,8,9,9,9,9,10,10,10,Cathode -,Anode +,Anode +,Cathode -,pH,pH,First dimension what is isoelectric focusing (IEF)?,How do you run IEF?,Separation Medium: IPG StripsInstrument: IEF Cell,IPG Strips,IPG stands for immobilized pH gradient Acrylamide gels are poured with a pH gradient onto a p
5、lastic backing, cut into strips and dehydrated pH gradients are created with sets of acrylamido buffers which are derivatives of acrylamide containing both reactive double bonds and buffering groups The pH gradient is fixed in the gel and doesnt change during focusing IPG strips allow for a high deg
6、ree of reproducibility and easy handling,IPG strips,IPG strips are available in a variety of pH gradients and lengths,ReadyStrip IPG strips,7 cm 11 cm 17 cm 18 cm 24 cm,Lengths,Broad range pH 3-10 pH 3-10 nonlinear (NL)Narrow Range pH 3-6 pH 4-7 pH 5-8 pH 7-10Micro Range pH 3.9-5.1 pH 4.7-5.9 pH 5.5
7、-6.7 pH 6.3-8.3,Broad Range pI Separations,pH 3-10,pH 3-10NL,IPG Strip Selection,pH 3-10,pH 3-6,pH 5-8,pH 7-10,E. coli Lysate,IPG Strip Resolution,Narrow,Micro,Broad,3-10,5-8,4.7-5.9,Sample/Rehydration buffer for IEF,ReadyPrep Rehydration/Sample Buffer 8 M urea, 2% CHAPS, 50 mM dithiothreitol (DTT),
8、 0.2% (w/v) Bio-Lyte 3/10 ampholytes, Bromophenol Blue问题： 1 加入这些目的是什么？ 2 可以有替代品吗？ / 为什么不加其他东西？（PCR反应体系）,Laemmli Sample Buffer for 1D SDS-PAGE,Why 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 SD
9、S-PAGE buffer and rehydration sample buffer?,Protein Solubility,加了一大堆东西，那么在什么情况下蛋白最容易溶解?Separate polypeptide chains如何能够达到这个目的呢？Breaking Molecular Interactions这些相互作用都包括什么呢？covalent interactions - disulfide bridgesnon-covalent interactions - ionic bonds, hydrogen bondshydrophobic interactions,Disrupti
10、on of Disulfide Bridges,Reducing agent 1 Mercaptoethanol - 700mM 碱性端的离子化，会破坏pH梯度2 Dithiothreitol (DTT) - 50mM 无离子化，但并非完美，较多的半胱氨酸二硫键难以被还原3 Tributylphosphine (TBP) - 2mM 挥发性、毒性、难闻气味、需要有机溶剂溶解，没有文献报道效果比DTT好,Disruption of Ionic Bond & Hydrogen Bond,Chaotrope 改变介电常数，氢键形成和极化（疏水键）几乎所有和蛋白质溶解相关的参数。 1 Urea 最主要
11、的功能是打破氢键，其次是离子键，而打破离子键是通过改变节电常数和蛋白质变性。 2 Thiourea 很强的致蛋白质变性作用，与Urea联合使用，明显促进蛋白质溶解。 问题：可以单独使用吗？,Disruption of Hydrophobic Interactions,Detergent： 1 Ionic - SDS 完全不能用吗？2 Nonionic - Triton, tween, NP-40, Brij, Mega 能用吗？3 Zwitterionic - CHAPS, ASB 能用吗？由于Urea的原因，在高浓度Urea情况下，CHAPS, Triton, NP-40可以使用。,Samp
12、le/Rehydration buffer for IEF,Isoelectric focusing rehydration & sample loading,Passive Rehydration: no voltage, sample is in rehydration buffer,Active Rehydration: low voltage, sample is in rehydration buffer,Cup Loading: rehydrate with buffer, add sample to cup with low voltage,Pros and cons of di
13、fferent rehydration techniques,Power conditions/programming,Strips should always be run at the highest voltage compatible with the heat dissipation capacities of the cell Volt hours: time integral of applied voltage. A standard for reproducing focusing conditions. This needs to be determined empiric
14、ally When an electrical field is applied to an IPG strip at the beginning of a run, the current will be high because of the high number of charged carriers present, as the proteins and ampholytes migrate, the current will gradually decrease because of decreasing charge Similar strips and samples should be run in batches so that the electrical conditions will be as consistent as possible Voltage is limited by current. If the maximum current is reached before the maximum voltage, the voltage will not reach the maximum,Questions?,
