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Sep 14, 2011 220 likes | 671 Views Download Presentation protein purification An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher. Presentation Transcript 1. PROTEIN PURIFICATION Step 1:Crude Extract 3. Overview OfThis Purification Making the ExtractPurification StepsAmmonium sulfate precipitation (salting out)DialysisIon Exchange Chromatography Each of these steps is a separate lecture. Each of these steps is a separate lecture. 4. Overview OfThis Purification Cont… Analysis and Verification Assays for specific activityPolyacrylamide gel electrophoresisWestern Blotting 5. Making The Extract Homogenize the source materialGoal: get target protein into solutionSource material Any biological material which contains abundant source of target proteinIf you’re lucky…Target protein is secreted outside the cellMost proteins found inside cell 6. Cell Disruption EnzymaticMechanicalGrindingHomogenizingMotorized or notHigh pressureSonication 8. Enzymatic MethodBug Buster™ from Novagen 11. Sonication Uses high frequency sound waves to break open bacteriaGenerates heat Need ethanol ice bathBuffer is protective 12. Avestin EmusiflexC5 Can process 160 liters 13. Two ImportantThings Method to assay proteinMethod to stabilize protein during the purification 14. Proteases Cell lysis during cell disruption releases proteases from cellular compartment (lysosomes)How do we keep them from destroying target protein?Keep cold (4°C)Some protocols add protease inhibitors 15. Choice OfExtraction Buffer Buffering pHBased on pH stability range of proteinIonic strengthDivalent cations 16. Choice OfExtraction Buffer Reducing agentsProtease inhibitorsReference and resource:Seidman and Moore 17. After Cell Disruption Centrifuge the homogenate to create the CRUDE EXTRACTTake aliquotsStore at 4°CSave the pellet (Why?) 18. Components OfLaboratory Solutions BuffersDifferent pH optimaUsually pH 6-8 19. SaltsIonic strengthAffects 3-D structureAffects solubility Because it affects electrostatic interactions between side chainsMg, Zn, Fe 21. Components OfLaboratory Solutions Divalent cationsCa, MgReducing agentsDithiothreitol, ß-mercaptoethanolProtease inhibitorsLeupeptinPMSFEtc. 22. To Learn More…. Protein Analysis and PurificationIan M RosenbergReference and resource:Seidman and MooreMany web resources

Purified proteins are required for many experimental applications, including structural studies and in vitro biochemical assays. Proteins can be obtained from a tissue or, more often, by their overexpression in a model organism, such as bacteria, yeast, or mammalian cells in culture. Protein purification involves isolating proteins from the source, based on differences in their physical properties. The objective of a protein purification scheme is to retain the largest amount of the functional protein with fewest contaminants. The purification scheme of a protein must be optimized to complete this process in the least number of steps. Figure 1. A Tris-buffered solution contains Tris base and its conjugate acid. The pKa of Tris at 25°C is 8.06, indicating that at pH = 8.06, 50% of the Tris is protonated (in its acidic form) and 50% is deprotonated (in its basic form). The article reviews four types of column chromatography that are commonly used in protein purification and discusses the advantages, disadvantages, and potential problems of each. This article also reports a Labome survey on 98 publications. The survey indicates that affinity column chromatography, mainly that based on HIS, GST, and FLAG tags, and size exclusion chromatography are the main methods cited in the publications. GE Healthcare is the major supplier of reagents and instruments used in protein purification. Developing a Protein Purification SchemeThe most important consideration in the development of a protein purification scheme is the downstream application of the purified protein. Both the quantity and purity of the protein must be sufficient for experimental analysis. Additionally, information about the behavior of the protein must be taken into consideration, as well-folded and functional protein is required for downstream studies. During purification and subsequent storage, many processes can occur that affect protein quality: protein unfolding, aggregation, degradation, and loss of function. Careful planning to purify protein as quickly as possible and under the most stabilizing conditions will maximize the chance of a successful purification scheme. Figure 2. An Aktaprime plus system for the automated chromatographic separation of proteins. From GE. Buffering componentThe solution conditions of a protein at each step of the

Their solubilization from the lipid bilayer while retaining their functional integrity. The typical approach involves the isolation of intracellular membranes by centrifugation followed by detergent solubilization of integral membrane proteins and high-speed centrifugation to remove insoluble membrane residues [55-57]. A wide range of detergents have been employed for membrane protein solubilization [58-60] and, in the absence of any literature or laboratory precedent, the investigator will need to determine the best detergent for their particular protein empirically. The solubilized membrane protein may then be purified by column chromatography in essentially the same way as for soluble proteins. However, the purification buffers will need to contain detergents to maintain the protein in a soluble state [61, 62]. Purification of membrane proteins is often extremely challenging due to loss of protein functional integrity and aggregation following initial removal from the lipid bilayer and through the various purification steps. Nonetheless, several groups have successfully purified membrane proteins in sufficient quantities for structure determination (see for example, Structural Genomics Consortium). Recently, nanodiscs have been successfully employed in the affinity purification of a Family B GPCR (see Labome article on nanodiscs) [63].Labome Survey of the Literature Citing Protein PurificationLabome surveyed publications citing protein purification methods. Table 10 lists the major suppliers and purification methods. Affinity and size exclusion methods are the most commonly used approaches in the literature. GE Healthcare is the predominant supplier for all methods, and Qiagen is the significant provider of HIS tag-based protein purification, and MilliporeSigma, of FLAG-based tag. typesuppliermajor brandnumsample referencesaffinityHISQiagenNi-NTA agarose/resin63 [64, 65] GE HealthcareNi Sepharose, HisTrap FF/HP37 [66, 67] ClontechTALON metal affinity23 [68, 69] Thermo Fisher / InvitrogenHisPur Cobalt/Ni-NTA resin9 [70, 71] Roche/ MilliporeSigmacOmplete His-Tag Purification Resins1 [72] Cube BiotechNi-NTA resin1 [73] GSTGE Healthcareglutathione Sepharose 4B66 [66, 74] MilliporeSigmaglutathione agarose3 [75, 76] FLAGMilliporeSigmaFLAG M2 affinity17 [77, 78] DNA binding proteinGE Healthcareheparin16 [67] maltose-binding protein / MBPNew England Biolabsamylose resin10 [73, 79] Other cited affinity-based protein purification systems include Strep-tactin resin from IBA Lifesciences for SBP-fused proteins [73], Streptactin Sepharose® High Performance from GE Healthcare (28-9355-99) [80]. size exclusionGE-HealthcareSuperdex / Superose / Sephacryl104 [81] ion exchangeanionGE HealthcareMono Q column11 [79,