蛋白质分离纯化How to detect, analyze and prepare?Different purposes, different protocols Detective/analytical level: nanogram(ng) or picogram(pg)Preparative level: milligram (mg)Protein detection/analysis:1, Hybridization: Western blot2, Immunological Techniques: Elisa & Co-IP3, electrophoresisSDS PAGE, 2-D gel, CE, IEF4, chromatography: HPLC, FPLC,5, Spectrometry: DLS, NMR, Mass-Spect, LC-MS 6, X-ray crystallography…Western blot: Protein-proteinThis method is dependent on the use of a high-quality antibody directed against a desired protein.Southern blot: DNA-DNA (Edward Southern. Detection of specific sequences among DNA fragments separated by gel electrophoresis.J Mol Biol. 1975 Nov 5;98(3):503-17)Northern blot: DNA-RNASDS-PAGE (sodium dodecyl(lauryl) sulfate-polyacrylamide gel electrophoresis)has a number of uses, which include:•Establishing protein size•Protein identification•Determining sample purity•Identifying disulfide bonds•Quantifying proteins•Blotting applicationsN-terminal protein sequencing by Edman chemistry 1.Samples can be supplied in solution or as membrane blots with more than 90% purities(μg level)2.If the sample is not pure enough, it might be separated with SDS-PAGE or 2-D gel electrophoresis and then electrotransfered to ProBlottTM PVDF membrane3.The length limit of the sequence obtained depends on the sample amount, the protein properties etc, ranging from 30 to 70 residues.pI: IEF (Isoelectric focusing)Focusing separation of species in an inhomogeneous medium (pH-gradient) according to their isoelectric points (pI).Molecular weight:SDS PAGE (polyacrylamide gel electrophoresis) HPLC (high pressure liquid chromatography)MS (Mass spectrometry)Protein concentration detection:BSA methodCo-efficientYeast ORF Ygr271c-apMDANTLNVSFEEILKGKKLDEDSIGLTLSP DKDHEDGSQVSPTQDRKELDQVVGEDEK DDFFENo W or Y, extinction coefficient is 0Absorption spectra of the aromatic amino acids in the near-ultraviolet regionFrom D. Wetlaufer, Adv. Protein Chem. (1962) 17:303-390.©1962 Academic PressUV detectionWhat is chromatography (色谱)?it is a broad range of physical methods used to separate and/or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase (固定相) bed and a mobile phase (流动相)which percolates (filters) through the stationary bed.How Does It Work?A mixture of various components enters a chromatography process, and the different components are flushed through the system at different rates.These differential rates of migration as the mixture moves over adsorptive materials provide separation. Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column.Liquid Chromatography1.reverse phase,2.high performance and3.size exclusionProtein surface propertiesReverse phase chromatography (RPC)Based on the hydrophobic interactions between solute (溶质) molecules and immobilized, matrix-bound ligands.RPC has found favour in a wide variety of preparative applications including micropurification of protein fragments for sequencing and process-scale purification of recombinant protein products.RPC also offers the benefit of exceptional flexibility in separation conditions so that either molecules of interest can be bound while contaminants pass through, or contaminants can be bound while the molecule of interest passes through.Reversed phase chromatography (RPC)Binding of the protein in a polar mobile phaseElution by changing the composition of the mobile phase to becomemore non-polarHydrophobic interaction chromatography (HIC)is a technique for the purification and separation of biomolecules based on differences in their surface hydrophobicity.Many biomolecules, generally considered to be hydrophilic, also have sufficient numbers of hydrophobic groups allowing interaction with hydrophobic ligands coupled to the chromatographic matrix.HIC vs RPC–Less substituted matrix –Less hydrophobic ligands–Weaker binding–Elution with water/dilute buffers–Native protein–Adsorption chromatography–Low pressure chromatography –More substituted matrix –More hydrophobic ligands–Stronger binding–Elution with non-polar solvents–Denatured protein–Partition chromatography–High performance liquid chromatography (HPLC)HIC RPCHigh performance liquid chromatography (HPLC)the process is conducted at a high velocity and pressure drop. The column is shorter and has a small diameter, but it is equivalent to possessing a large number of equilibrium stages.Size exclusion chromatography, also known as gel permeation or filtration chromatography (分子筛)does not involve any adsorption and is extremely fast (FPLC).The packing is a porous gel, and is capable of separating large molecules from smaller ones.The larger molecules elute first since they cannot penetrate the pores. This method is common in protein separation and purification.Ion Exchange Chromatography(离子交换层析)Ion exchange chromatography is commonly used in the purification of biological materials.There are two types of exchange:cation exchange (S)in which the stationary phase carries a negative charge, and anion exchange (Q)in which the stationary phase carries a positive charge.An increase in ionic strength is typically used to elute proteins from IEX media. However, it is possible to use a change in pH to displace bound material.Affinity Chromatography(亲和层析)involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds.The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or "affinity tails/tag", that are inserted into the matrix can be genetically engineered to possess a specific affinity.GKPIPNPLLGLDST V5MASMTGGQQMG T7EQKLISEEDL MycProtien Maltose Binding Protein (MBP)HHHHHH Poly-HisYPYDVPDYA HAProtein GSTEYMPME GLU-GLUProteinGFP (Green Fluorescent Protein)KRRWKKNFIAVSAANRFKKISSSGAL Calmodulin Binding ProteinEpitope TagSequenceThe two strategies:1, fish out the target protein from the mix 2, remove the contaminants from the target proteinPurification by removing the target molecule from the contaminants.Affinity chromatography techniques are very specific for the target molecule or for a group of molecules with closely related biological properties. This makes them capable of "fishing out" the target molecule (or the group), leaving all contaminants behind.When applicable these techniques are to be preferred, since they drastically simplify the purification protocol.Purification by removing the contaminants from the target molecule.When a suitable affinity chromatography technique is not at hand, one has to rely on a sequence of general chromatography techniques to remove the contaminants.A typical purification protocol when nothing is known about the target protein employs the IEX-HIC-GF sequence of purification steps.蛋白质的理化性质及其分离策略的选择：1.分子大小2.形状3.溶解度4.电荷5.疏水性6.密度7.亲和能力8.可逆性缔合9.稳定性10.表面活性1.分子大小1.1 透析和超滤1.2 离心、密度梯度离心1.3 凝胶过滤2. 形状球状蛋白具有较小的有效半径(斯托克半径)膜过滤凝胶过滤3. 溶解度等电点沉淀盐溶(NaCl)和盐析(NH4)2 SO4有机溶剂(PEG, Ethanol)分级4. 电荷电泳(pI: 0.02 pH unit)离子交换5. 疏水性蛋白质表面的疏水氨基酸残基的数目和分布6. 密度一般1.3-1.4 g/ml含大量磷酸盐或脂质的蛋白质不同7. 亲和能力重组融合蛋白(GST-, His-tag et al.)配基(底物、抑制剂、辅因子、抗体)8. 可逆性缔合在不同条件下，蛋白质的聚合状态可能不同9. 稳定性热稳定性酶解稳定性10. 表面活性泡沫分离反胶团相转移聚合物-盐-水液-固萃取体系。