蛋白質(zhì)的分離與純

1、純化目的純化目的: 研究其結(jié)構(gòu)、組成、性質(zhì)和功能等。研究其結(jié)構(gòu)、組成、性質(zhì)和功能等。 純化方法的依據(jù)純化方法的依據(jù)：蛋白質(zhì)的基本性質(zhì)：蛋白質(zhì)的基本性質(zhì) 1）溶解性：鹽析等。）溶解性：鹽析等。 2）分子大小和形狀：凝膠過(guò)濾（分子篩過(guò)濾），）分子大小和形狀：凝膠過(guò)濾（分子篩過(guò)濾），透析，超離心，超濾等。透析，超離心，超濾等。 3）電荷（酸堿性質(zhì)）：電泳，離子交換層析，等）電荷（酸堿性質(zhì)）：電泳，離子交換層析，等電聚焦等。電聚焦等。 4）吸附性質(zhì)：吸附層析，疏水互作層析。）吸附性質(zhì)：吸附層析，疏水互作層析。 5）特異結(jié)合親和性：親和層析。）特異結(jié)合親和性：親和層析。 有的方法利用了蛋白質(zhì)幾個(gè)方面的性

2、質(zhì)。有的方法利用了蛋白質(zhì)幾個(gè)方面的性質(zhì)。 一般要連續(xù)采用多種方法才能達(dá)到純化的目一般要連續(xù)采用多種方法才能達(dá)到純化的目的。的。Stabilization of proteins 蛋白質(zhì)的穩(wěn)定蛋白質(zhì)的穩(wěn)定 防止蛋白質(zhì)變性或失活。防止蛋白質(zhì)變性或失活。 措施：措施： 1）緩沖）緩沖pH； 2）低溫；）低溫；250C,一般一般40C; 3) 加蛋白酶抑制劑加蛋白酶抑制劑;Assay of proteins 蛋白質(zhì)的檢測(cè)蛋白質(zhì)的檢測(cè) 純化過(guò)程中純化過(guò)程中,不斷檢測(cè)目的蛋白質(zhì)的存在和不斷檢測(cè)目的蛋白質(zhì)的存在和純度變化。純度變化。 酶：檢測(cè)其催化產(chǎn)生的產(chǎn)物。酶：檢測(cè)其催化產(chǎn)生的產(chǎn)物。 非酶蛋白質(zhì)：檢測(cè)其生

3、物學(xué)效應(yīng)，如抗原抗非酶蛋白質(zhì)：檢測(cè)其生物學(xué)效應(yīng)，如抗原抗體反應(yīng)。體反應(yīng)。（一）鹽析：（一）鹽析：在蛋白質(zhì)溶液中加入大量中性鹽，以破壞蛋白質(zhì)的在蛋白質(zhì)溶液中加入大量中性鹽，以破壞蛋白質(zhì)的膠體性質(zhì)，使蛋白質(zhì)從溶液中沉淀析出，稱(chēng)為膠體性質(zhì)，使蛋白質(zhì)從溶液中沉淀析出，稱(chēng)為鹽析鹽析。依據(jù)：大多數(shù)蛋白質(zhì)在高鹽濃度時(shí)溶解度降低。依據(jù)：大多數(shù)蛋白質(zhì)在高鹽濃度時(shí)溶解度降低。 鹽析對(duì)樣品有濃縮作用。鹽析對(duì)樣品有濃縮作用。溶液中加入一定量的中性鹽如溶液中加入一定量的中性鹽如NHNH4 4 2 2SOSO4 4A A 由于由于 NHNH4 4 2 2SOSO4 4的親水性比蛋白質(zhì)親水性大，能與大的親水性比蛋白質(zhì)親水性

4、大，能與大量的水分子結(jié)合，使量的水分子結(jié)合，使蛋白質(zhì)表面蛋白質(zhì)表面水膜退化、消失；水膜退化、消失；B NH42SO4解離，中和了蛋白質(zhì)顆粒所帶的電荷，破壞解離，中和了蛋白質(zhì)顆粒所帶的電荷，破壞蛋白質(zhì)表面電荷層而沉淀析出蛋白質(zhì)表面電荷層而沉淀析出 Size-exclusion chromatography,also called gel filtration, separates proteins according to size. Thecolumn matrix is a cross-linked polymer with pores of selected size.Larger pro

5、teins migrate faster than smaller ones, because they are toolarge to enter the pores in the beads and hence take a more directroute through the column. The smaller proteins enter the pores andare slowed by their more labyrinthine path through the column.Ion-exchange chromatography exploits differenc

6、es in the sign and magnitude of the net electric charges of proteins at a given pH. The column matrix is a synthetic polymer containing bound charged groups; those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion exchangers. Ion-exchange c

7、hromatography on a cation exchanger is shown here. The affinity of each protein for the charged groups on the column is affected by the pH (which determines the ionization state of the molecule) and the concentration of competing free salt ions in the surrounding solution. Separation can be optimize

8、d by gradually changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient.(c) Affinity chromatography separates proteins by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads

9、. (In biochemistry, the term “l(fā)igand” is used to refer to a group or molecule that binds to a macromolecule such as a protein.) After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted (washed out of the column) by a solution con

10、taining free ligand.特點(diǎn)特點(diǎn):1)1)使用的固定相支持劑顆粒很細(xì)使用的固定相支持劑顆粒很細(xì), ,表面積很大表面積很大.2).2)溶劑系統(tǒng)采取高壓溶劑系統(tǒng)采取高壓, ,洗脫速度增大洗脫速度增大.3).3)高分辨率高分辨率反相反相HPLC(reversed-phase HPLC):HPLC(reversed-phase HPLC):固定相是非極固定相是非極性的烷基硅烷與硅膠形成的鍵合相，與被分離物只性的烷基硅烷與硅膠形成的鍵合相，與被分離物只可能有疏水作用；流動(dòng)相是相對(duì)極性的可能有疏水作用；流動(dòng)相是相對(duì)極性的High performance liquid chromatogra

11、phy 高效液相層析高效液相層析(HPLC)(1)A modern refinement in chromatographic methods is HPLC, or high-performance liquid chromatography. HPLC makes use of high-pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can withstand t

12、he crushing force of the pressurized flow. By reducing the transit time on the column, HPLC can limit diffusional spreading of protein bands and thus greatly improve resolution. Electrophoresis.Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection current