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  蛋白質純化簡介(Translated by ABDC)
Protein purification
From Wikipedia, the free encyclopedia http://en.wikipedia.org/wiki/Protein_purification

Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture.
Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest.
The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins.
Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps may exploit differences in (for example) protein size, physico-chemical properties, binding affinity and biological activity.

蛋白質純化 蛋白質純化是要從一個複雜的混合物中分離出一個單一類型蛋白質的一系列處理程序.
蛋白質的純化對於蛋白質的功能, 結構和交互作用等的特性的確認是非常重要的.
初始材料通常是生物組織或微生物的培養體.
純化程序的各個步驟的, 可能包括 從局限它的母體中釋放出蛋白質, 從混合物中分離蛋白質和非蛋白質部分, 最後是從所有其他蛋白質中分離出所要的蛋白質.
從所有其他蛋白質中分離出一種蛋白質, 通常是最費力的蛋白質純化的面向. 分離步驟可以利用例如蛋白質的大小, 物理-化學性質, 結合親和力和生物活性等差異.
Contents 內容
1 Purpose 目的
2 Strategies 策略
3 Evaluating purification yield 評估提純產量
4 Methods of protein purification 蛋白質純化的方法
5 Extraction 萃取
6 Precipitation and differential solubilization 沉澱和差溶
7 Ultracentrifugation 超速離心
8 Chromatographic methods 層析方法
8.1 Size exclusion chromatography 斥濾層析法
8.2 Separation based on charge or hydrophobicity
      基於電荷或疏水性的分離
8.3 Ion exchange chromatography 離子交換層析法
8.4 Affinity chromatography 親和層析
8.4.1 Metal binding 金屬結合
8.4.2 Immunoaffinity chromatography 免疫親和層析法
8.4.3 Purification of a tagged protein 標記的蛋白質的純化
8.5 HPLC 高效液相層析法
9 Concentration of the purified protein 純化的蛋白質的濃縮
9.1 Lyophilization 冷凍乾燥
9.2 Ultrafiltration 超過濾
10 Analytical 分析(目的的)
10.1 Denaturing-Condition Electrophoresis 變性條件電泳
10.2 Non-Denaturing-Condition Electrophoresis 非變性條件電泳 11 References 參考
12 External links 外部鏈接

Purpose 目的
Purification may be preparative or analytical.
Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use. Examples include the preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin).
Analytical purification produces a relatively small amount of a protein for a variety of research or analytical purposes, including identification, quantification, and studies of the protein's structure, post-translational modifications and function.
Pepsin and urease were the first proteins purified to the point that they could be crystallized.[1]

純化的可以是以製備或分析為目的.
製備的純化目標是產生一個相對比較大量的純化蛋白質, 以供後續使用.
例子包括商業產品的製備, 如酶(乳糖酶) , 營養蛋白(如大豆蛋白分離物), 某些生物製藥(如胰島素).
以分析目的純化, 產生一個相對比較少量的蛋白質, 用於各式各樣的研究或分析的目的, 包括定性, 定量, 和蛋白質的結構, 轉譯後修飾和功能等的研究.
胃蛋白酶和脲酶是第一個被純化到可以被結晶的程度的蛋白質.

Strategies 策略
Recombinant bacteria can be grown in a flask containing growth media.  Choice of a starting material is key to the design of a purification process.
In a plant or animal, a particular protein usually isn't distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein.
Use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein.
If the protein is present in low abundance, or if it has a high value, scientists may use recombinant DNA technology to develop cells that will produce large quantities of the desired protein(this is known as an expression system).
Recombinant expression allows the protein to be tagged, e.g. by a His-tag, to facilitate purification, which means that the purification can be done in fewer steps. In addition, recombinant expression usually starts with a higher fraction of the desired protein than is present in a natural source.

基因重組細菌可以生長在含有的生長介質燒瓶中.
起始原料的選擇是純化過程的設計關鍵.
在一種植物或動物中, 一種特定的蛋白質通常是沒有均勻地分佈在整個身體, 不同的器官或組織有高一些或低一些濃度的蛋白質. 只有使用濃度最高的組織或器官, 減少產生一個給定量的純化蛋白質所需的體積.
如果蛋白質存的含量是低的, 或者如果它具有很高的價值, 科學家可能利用DNA重組技術以開發能產生大量所需蛋白質的細胞. (這就是所謂的表達系統). 重組表達允許蛋白質被標記, 例如經由His-標記, 便於純化, 這表示的純化可以以更少的步驟來完成. 此外, 重組表達通常以比存在於天然來源更高組成含量的所需蛋白質開始.

An analytical purification generally utilizes three properties to separate proteins.
First, proteins may be purified according to their isoelectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis.

Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity.
This is very useful for scientific purposes and the detection limits for protein are nowadays very low and nanogram amounts of protein are sufficient for their analysis.

Thirdly, proteins may be separated by polarity/hydrophobicity via high performance liquid chromatography or reversed-phase chromatography.

以分析為目的純化, 通常採用三個性質以分離蛋白質.
首先, 蛋白質可依據其不同的等電點, 通過pH梯度凝膠或離子交換分離管柱被純化.
其次, 蛋白質可依據其不同的等電點, 經由斥濾層析法或 SDS-PAGE分析法(十二烷基硫酸鈉 - 聚丙烯酰胺凝膠電泳)被分離.
蛋白質也常常經由使用2D-PAGE法純化, 然後以肽質譜指紋分析以建立蛋白質的成份定性. 這對於科學目的非常有用, 且其現今的檢測限是非常低, 用於科學目的,這是非常有用, 且蛋白質的檢測限是時下非常低的, 毫微克量的蛋白質是足以滿足他們的分析的.
第三, 經由高效液相層析法或反相層析法, 蛋白質可以其極性/疏水性被分開.

Evaluating purification yield 評估提純產量
The most general method to monitor the purification process is by running a SDS-PAGE of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar apparent molecular weight.

監測純化過程最通常的方法是對不同步驟, 加以進行的SDS-PAGE分析. 這個方法僅得出混合物中不同蛋白質含量的一個粗略的衡量, 它是不能夠區表面上分子量分相似的蛋白質的.

If the protein has a distinguishing spectroscopic feature or an enzymatic activity, this property can be used to detect and quantify the specific protein, and thus to select the fractions of the separation, that contains the protein.
If antibodies against the protein are available then western blotting and ELISA can specifically detect and quantify the amount of desired protein.
Some proteins function as receptors and can be detected during purification steps by a ligand binding assay, often using a radioactive ligand.

如果該蛋白具有一個顯著的光譜特徵或酵素的活性, 此性質可用於檢測和定量該特定的蛋白質, 並由此選別出含有該蛋白質的分離物的部份.
如果針對該蛋白質的抗體是可以取得的, 則免疫印跡和ELISA(酵素免疫分析法) 即可以專一地檢測和定量目標蛋白質.
一些蛋白質有受體的功能, 並在純化過程的步驟中, 以配體連結分析法被測定,  通常使用放射性配體.

In order to evaluate the process of multistep purification, the amount of the specific protein has to be compared to the amount of total protein.
The latter can be determined by the Bradford total protein assay or by absorbance of light at 280 nm, however some reagents used during the purification process may interfere with the quantification. For example, imidazole (commonly used for purification of polyhistidine-tagged recombinant proteins) is an amino acid analogue and at low concentrations will interfere with the bicinchoninic acid (BCA) assay for total protein quantification.

Impurities in low-grade imidazole will also absorb at 280 nm, resulting in an inaccurate reading of protein concentration from UV absorbance.

為了評估多步驟純化程序, 特定蛋白質的量需與總蛋白量進行比較. 後者可以Bradford總蛋白分析法或以280nm波長的吸光度測定, 不過, 一些在純化過程中所使用的試劑可能會干擾其定量. 例如,咪唑(通常用於純化以多聚組氨酸作標記的重組蛋白)是一種氨基酸類似物, 其在低濃度時, 將干擾與二辛可寧酸(BCA)分析法的總蛋白定量.
低等級咪唑中的雜質在280nm處也會吸收, 造成由UV吸收所得到的不準確蛋白質濃度讀值.

Another method to be considered is Surface Plasmon Resonance (SPR). SPR can detect binding of label free molecules on the surface of a chip.
If the desired protein is an antibody, binding can be translated directly to the activity of the protein.
One can express the active concentration of the protein as the percent of the total protein.
SPR can be a powerful method for quickly determining protein activity and overall yield. It is a powerful technology that requires an instrument to perform.

另外一種被考慮到的方法是 表面等離子體共振(SPR). SPR可以檢測連結在晶片上的無標記分子. 如果所要的蛋白質是抗體, 其連結可以被直接解讀為蛋白質的活性. 蛋白質的活性濃度的表示方式之一為總蛋白的百分比.
SPR可以是一個快速測定蛋白活性和總產率的強大的方法. 這是需要儀器來執行的一個功能強大的技術.

Methods of protein purification 蛋白質純化的方法

The methods used in protein purification can roughly be divided into analytical and preparative methods.
The distinction is not exact, but the deciding factor is the amount of protein that can practically be purified with that method.
Analytical methods aim to detect and identify a protein in a mixture, whereas preparative methods aim to produce large quantities of the protein for other purposes, such as structural biology or industrial use.
In general, the preparative methods can be used in analytical applications, but not the other way around.

在蛋白質純化中使用的方法大致可以分為分析目的和製備目的的方法.
其區別是不精確的, 但是決定性的因素是使用該方法的所能實際被純化的蛋白質的量.
以分析為目的方法目標在於進行檢測和識別混合物中的蛋白質, 而製備方法目標在於製造大量用於其他目的蛋白質, 如結構生物學或工業使用.
一般情況下, 製備方法可以在分析的應用中使用的, 但並非反之亦然.

Extraction 萃取
Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it.
There are several methods to achieve this: Repeated freezing and thawing, sonication, homogenization by high pressure, filtration, or permeabilization by organic solvents.
The method of choice depends on how fragile the protein is and how sturdy the cells are. After this extraction process soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by centrifugation.
The extraction process also extracts proteases, which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis.

依來源而定, 必須破壞含蛋白質的組織或細胞, 將其帶到成溶液中.
有幾種方法來達成: 反復凍融, 超音波處理, 用高壓均質化, 過濾, 或以有機溶劑浸透.
方法的選擇取決於蛋白有多麼脆弱, 以及細胞有多堅固. 在此提取過程之後, 可溶性蛋白質的將在該溶劑中, 並可以以離心方式與細胞膜,DNA等分離.
此提取過程中也會提取蛋白酶, 它在溶液中就以開始在消化蛋白質了. 如果該蛋白質對於蛋白水解作用是敏感的, 它通常是期望能迅速地進行, 並保持提取液冷卻, 以減緩蛋白水解.

Precipitation and differential solubilization 沉澱和差溶
Main article: Ammonium sulfate precipitation
In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH4)2SO4. This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitate protein. Ammonium sulphate can be removed by dialysis.
The hydrophobic groups on the proteins gets exposed to the atmosphere and it attracts other protein hydrophobic groups and gets aggregated. Protein precipitated will be large enough to be visible. One advantage of this method is that it can be performed inexpensively with very large volumes.

硫酸銨沉澱
在大量的蛋白質純化中,一個共同的分離蛋白質的第一步是以硫酸銨(NH4)2SO4沉澱. 這是經由加入漸增量的硫酸銨的, 並收集不同部份的蛋白質沉澱. 硫酸銨可以用透析法除去.
蛋白質上的疏水性基團被暴露在大氣中, 它吸引了其他蛋白質的疏水性基團, 因而凝集起來. 蛋白質沉澱將大到看得見. 這種方法的優點之一是它可以非常大量廉價地進行.

The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in order to isolate any one particular protein from others that are in the same membrane compartment.
Sometimes a particular membrane fraction can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane.
A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein's native conformation during complete purification.

最先被純化的蛋白質是水溶性蛋白質.
完整的膜蛋白的純化需要破壞細胞膜, 以能夠在與其他蛋白質相同的膜隔室中, 分離出任何一個特定的蛋白質. 有時一個特定的膜的部份可以先被分離出來, 例如在純化位於線粒體膜的蛋白質之前, 從細胞先分離線粒體. 如十二烷基硫酸鈉(SDS)的洗滌劑, 可以用來溶解細胞膜, 使在純化過程中的膜蛋白保持在溶液中.
然而,因為SDS引起變性, 較溫和的清潔劑, 如Triton X-100或CHAPS, 可用於在完整的純化過程中, 保留蛋白質的天然構型.

Ultracentrifugation 超速離心
Main article: Ultracentrifuge Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid.
When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the inertia of each particle yields an outward force proportional to its mass.
The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liquid exerts on the particle.
The net effect of "spinning" the sample in a centrifuge is that massive, small, and dense particles move outward faster than less massive particles or particles with more "drag" in the liquid. When suspensions of particles are "spun" in a centrifuge, a "pellet" may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid.

超速離心
離心分離是一個利用離心力分離懸浮在液體中的不同質量或密度顆粒的混合物的過程. 當容器(通常是一個管或瓶) 中含有的蛋白質或其他顆粒物的混合物, 如細菌細胞, 是在高速下旋轉時, 每個粒子的慣性產生一個向外的力, 與它的質量成正比.
一個粒子因為這個力而移動通過液體的傾向, 會被液體施加在粒子的阻力抵減了. 樣品在離心操作的淨效應是塊狀, 小, 緻密顆粒, 比起比質量較小的粒子或在液體中的較會"拖" 的顆粒, 更快地向外移動. 當懸浮液顆粒在離心中的"旋轉", "團顆粒”就可能在容器底部形成, 其中富含液體中具有低阻力的最龐大顆粒.

Non-compacted particles remain mostly in the liquid called "supernatant" and can be removed from the vessel thereby separating the supernatant from the pellet.
The rate of centrifugation is determined by the angular acceleration applied to the sample, typically measured in comparison to the g.
If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the particles accumulate specifically at a point in the vessel where their buoyant density is balanced with centrifugal force. Such an "equilibrium" centrifugation can allow extensive purification of a given particle.

沒有被壓緊的顆粒大部份是留在稱為"上層澄清液" 的液體裏, 並可以從容器中取出, 因而將"上層澄清液" 與 "團顆粒" 分離.
離心分離的速率是由施加到樣品的角加速度決定的, 通常以與重力加速度g 比較. 如果樣品離心的時間夠長, 在容器中的顆粒將達到平衡, 其中顆粒在容器中的一個特定的點積聚, 在此, 其浮力密度與離心力是平衡的. 這樣的 "平衡" 離心分離可以允許大量純化一個特定的粒子.

Sucrose gradient centrifugation — a linear concentration gradient of sugar (typically sucrose, glycerol, or a silica based density gradient media, like Percoll) is generated in a tube such that the highest concentration is on the bottom and lowest on top.
Percoll is a trademark owned by GE Healthcare companies.
A protein sample is then layered on top of the gradient and spun at high speeds in an ultracentrifuge. This causes heavy macromolecules to migrate towards the bottom of the tube faster than lighter material. During centrifugation in the absence of sucrose, as particles move farther and farther from the center of rotation, they experience more and more centrifugal force (the further they move, the faster they move).

蔗糖梯度離心 : 一種糖線性濃度梯度(通常是蔗糖, 甘油, 或基於二氧化矽的密度梯度介質, 如Percoll) 被製作在一個管中, 最高濃度是在底部和最低的在頂部. 珀是由GE醫療公司擁有的商標. 然後蛋白質樣品放置於梯度的上層, 然後在超速離心下的高速旋轉. 這導致重的大分子比較輕的材料更快地移向管的底部. 在在沒有蔗糖的離心過程中, 由於顆粒從離旋轉的中心越來越遠地移動, 它們的會受到越來越大的離心的力量(他們移動地越遠, 就移動的更快.

The problem with this is that the useful separation range of within the vessel is restricted to a small observable window. Spinning a sample twice as long doesn't mean the particle of interest will go twice as far, in fact, it will go significantly further.
However, when the proteins are moving through a sucrose gradient, they encounter liquid of increasing density and viscosity.
A properly designed sucrose gradient will counteract the increasing centrifugal force so the particles move in close proportion to the time they have been in the centrifugal field.
Samples separated by these gradients are referred to as "rate zonal" centrifugations.
After separating the protein/particles, the gradient is then fractionated and collected.

這種方法的問題是, 在容器內的有用的分離範圍被限制在一個小的可觀察窗口.
對一個樣品作2次旋轉, 並不意味著感興趣的顆粒將到兩倍遠之, 實際上, 它會明顯地到更遠些.
然而, 當將蛋白質通過蔗糖密度梯度, 它們所遇到的液體的密度和粘度漸增. 適當設計的蔗糖梯度將抵消漸增的離心力, 所以粒子移動與他們在離心場中的時間的比例很接近. 樣品在這些梯度中被分離被稱為 "速率區帶離心".
分離後的蛋白質/顆粒, 梯度等, 接著被分級分離和收集.

Chromatographic methods 層析方法

Chromatographic equipment. Here set up for a size exclusion chromatography. The buffer is pumped through the column (right) by a computer controlled device.
Usually a protein purification protocol contains one or more chromatographic steps.
The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials.
Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column.
Usually proteins are detected as they are coming off the column by their absorbance at 280 nm.
Many different chromatographic methods exist:

層析儀器. 這裡的設置是斥濾層析. 以計算機控制的設備使緩衝液由幫蒲輸送通過層析分離管(右).
通常, 一個蛋白質純化的方案包含一個或多個層析步驟. 層析法的基本步驟是使含有蛋白質的的溶液流過以各種材料填充的層析分離管. 不同的蛋白質與層析分離管填充的材料有不同的相互作用, 因此可以以通過分離管所需的時間來被分開, 或需要將蛋白質從層析分離管沖滌出來的條件. 通常是當蛋白質從層析分離管出來時, 由它們在280 nm波長的吸光度變化被檢出的.
有許多不同的層析方法存在.

Size exclusion chromatography 斥濾層析法
Main article: Gel permeation chromatography
Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels.
This technique is known as size exclusion chromatography.
The principle is that smaller molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of a certain range in size will require a variable volume of eluent (solvent) before being collected at the other end of the column of gel.
In the context of protein purification, the eluent is usually pooled in different test tubes. All test tubes containing no measurable trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins.

凝膠滲透層析法
層析法可以使用多孔性凝膠以分離在溶液中或變性條件下的蛋白質.
這種技術被稱為斥濾層析法. 其原理是, 較小的分子必須經過較大數量的多孔基質. 因結果是, 在一定尺寸範圍內的蛋白質, 在層析分離管的膠體的另一端被收集之前, 將需要一個可變體積的沖滌液(溶劑).
在蛋白質純化的前後關係中, 滌出物通常是在不同的試管中匯集. 所有不含有可測量微量蛋白質的試管都被丟棄. 剩餘的溶液中即是由要純化的蛋白質或其他類似大小的蛋白質組成的.

Separation based on charge or hydrophobicity
基於電荷或疏水性的分離
Hydrophobic Interaction Chromatography Resin used in the column are amphiphiles with both hydrophobic and hydrophilic regions.
The hydrophobic part of the resin attracts hydrophobic region on the proteins. The greater the hydrophobic region on the protein the stronger the attraction between the gel and that particular protein.

疏水交互作用層析 層析分離管中的所用的樹脂是具有疏水性和親水性區域的雙面親合物. 樹脂的疏水性部分吸引在蛋白質上的疏水性的區域. 蛋白質上的疏水區愈大, 在膠與特定的蛋白質之間的相互吸引力越強.

Ion exchange chromatography  離子交換層析法
Main article: Ion exchange chromatography
Ion exchange chromatography separates compounds according to the nature and degree of their ionic charge.
The column to be used is selected according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged compounds, while cation exchange resins have a negative charge and are used to separate positively charged molecules.

離子交換層析法
離子交換層析法根據其離子電荷的性質和程度分離化合物.
所要使用的層析分離管的選擇是根據它的類型和電荷強度. 陰離子交換樹脂帶有正電荷, 並用於保留和分離帶負電荷的化合物, 而陽離子交換樹脂帶有負電荷, 並用於分離帶正電荷的分子.

Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions.
Upon injection of the sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each solute depends upon the strength of its charge. The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the separation.

分離開始前緩衝溶液被幫蒲輸送通過層析分離管使電荷相反的離子達到平衡. 在注射樣品後之, 溶質分子會與緩衝溶液離子競爭在樹脂上的結合位點.
每個溶質滯留時間長度, 取決於其荷電的強度. 荷電最微弱的化合物將先沖滌出來, 接著的是那月愈來愈強荷電的. 由於分離機制的本質, pH值, 緩衝溶液類型, 緩衝液的濃度, 以及溫度等, 在分離的控制裏, 都扮演著重要的角色.

Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently used in both analytical and preparative separations. nickel-affinity column. The resin is blue since it has bound nickel.
用於蛋白質純化, 離子交換層析法是一個非常強大的工具, 是經常被使用於分析和製備分離. 鎳親和層析分離管. 因它已經被連結了鎳, 該樹脂是藍色的.

Affinity chromatography 親和層析
Main article: Affinity chromatography
Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands attached to their surfaces which are specific for the compounds to be separated.
Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions.
This "lock and key" fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the sample is unretained.

親和層析親和層析是一種基於根據分子構型的分離技術, 它經常使用特定應用所需的樹脂. 這些樹脂具有連接到其表面的配體, 它們對於所要被分離的化合物具有專一性.
最經常​​的是, 這些配體發揮了類似抗體 - 抗原相互作用的類似方式的功能. 這種在配體及其目標化合物之間的"鎖和鑰匙", 使得它高度的專一, 經常產生一個單峰, 而樣品中的其他所有成份都不被滯留.

Many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography.
Detergent-solubilized proteins can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin.
Proteins that do not bind to the lectin are washed away and then specifically bound glycoproteins can be eluted by adding a high concentration of a sugar that competes with the bound glycoproteins at the lectin binding site.
Some lectins have high affinity binding to oligosaccharides of glycoproteins that is hard to compete with sugars, and bound glycoproteins need to be released by denaturing the lectin.

許多膜蛋白是糖蛋白, 可以以凝集素親和層析法來存純化.
可溶解在清潔劑的蛋白質, 可以允許被連結到已被修改為含有一個共價鍵連接的凝集素層析樹脂上. 不會與凝集素連結的蛋白質會被被沖走, 然後具連結專一性的糖蛋白, 可經由添加高濃度, 在結合位點與糖蛋白糖競爭的一個糖類, 而被沖滌出來.
一些凝集素與糖蛋白寡醣部份具有高親和力連結, 很難用糖類去競爭, 需要使凝集素變性, 被連結的糖蛋白才能被釋放.

Metal binding 金屬結合
Main article: Polyhistidine-tag
A common technique involves engineering a sequence of 6 to 8 histidines into the N- or C-terminal of the protein.
The polyhistidine binds strongly to divalent metal ions such as nickel and cobalt. The protein can be passed through a column containing immobilized nickel ions, which binds the polyhistidine tag. All untagged proteins pass through the column.
The protein can be eluted with imidazole, which competes with the polyhistidine tag for binding to the column, or by a decrease in pH (typically to 4.5), which decreases the affinity of the tag for the resin. While this procedure is generally used for the purification of recombinant proteins with an engineered affinity tag (such as a 6xHis tag or Clontech's HAT tag), it can also be used for natural proteins with an inherent affinity for divalent cations.

組氨酸標記
一種常用技術涉及到將6至8個組氨酸序列工程化地連接到蛋白質N-或C-末端, 多聚組氨酸會與二價金屬離子, 如鎳和鈷強烈結合.
此蛋白質可被通過一含有固定化鎳離子的層析分離管, 它(鎳離子)將連結多聚組氨酸標記. 所有未標記的蛋白質會通過層析分離管.
此種蛋白質可以用咪唑沖滌出來, 它在層析分離管的連結位置, 與組氨酸標記競爭. 或經由pH值下降(通常為4.5), 降低標記對於樹脂的親合性.
雖然此程序通常用於具工程化(精心設計)親和標記的重組蛋白的純化, (如一個6xHis標記或Clontech公司的HAT標記), 它也可以用於對二價陽離子具有固有親和性的天然蛋白質上.

Immunoaffinity chromatography 免疫親和層析法
A HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced column and an apparatus for measuring the absorbance.
Main article: Immunoaffinity chromatography
Immunoaffinity chromatography uses the specific binding of an antibody to the target protein to selectively purify the protein.
The procedure involves immobilizing an antibody to a column material, which then selectively binds the protein, while everything else flows through.
The protein can be eluted by changing the pH or the salinity. Because this method does not involve engineering in a tag, it can be used for proteins from natural sources.[2]

一種高效液相層析. 由左到右:一幫蒲設備, 可產生兩種不同溶劑比例梯度, 一鋼強化的層析分離管, 一用於測定吸光度的裝置.
主要文章:免疫親和層析法
免疫親和層析法使用的一個抗體對於目標蛋白的特定連結, 以選擇性地純化此蛋白質.
此過程涉及到將抗體固定在層析分離管填料, 然後, 它(分離管填料)選擇性地結合此蛋白質, 其他的東西均流流過去.
蛋白質可以機經由改變pH值或鹽度被沖滌出來. 因為這個方法不涉及工程化(精心設計)的標記, 它可以用於天然來源的蛋白質.

Purification of a tagged protein 標記的蛋白質的純化
Another way to tag proteins is to engineer an antigen peptide tag onto the protein, and then purify the protein on a column or by incubating with a loose resin that is coated with an immobilized antibody.
This particular procedure is known as immunoprecipitation. Immunoprecipitation is quite capable of generating an extremely specific interaction which usually results in binding only the desired protein. The purified tagged proteins can then easily be separated from the other proteins in solution and later eluted back into clean solution.
When the tags are not needed anymore, they can be cleaved off by a protease. This often involves engineering a protease cleavage site between the tag and the protein.
標記蛋白質的另一種方法是工程化(精心設計)一個具抗原特性的肽標記到蛋白質上, 然後在塗有一種固定化的抗體的層析分離管或鬆散的樹脂溫育來進行純化.
這種特殊的過程被稱為免疫沉澱. 免疫沉澱相當程度地, 能夠產生一個非常專一性的交互作用 , 這通常能導致只結合有所需的蛋白質. 已純化的被標記蛋白質, 接著,就可以在溶液中很容易地被從其他蛋白質分離, 之後再沖滌回到乾淨的溶液中.
當標記不再需要的了, 它們能被蛋白酶裂解掉. 這經常涉及到在標記和蛋白質之間, 工程化(精心設計)一個蛋白酶裂解位點.

HPLC 高效液相層析法
Main article: High performance liquid chromatography
High performance liquid chromatography or high pressure liquid chromatography is a form of chromatography applying high pressure to drive the solutes through the column faster.
This means that the diffusion is limited and the resolution is improved. The most common form is "reversed phase" hplc, where the column material is hydrophobic.
The proteins are eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity.
After purification by HPLC the protein is in a solution that only contains volatile compounds, and can easily be lyophilized.[3]
HPLC purification frequently results in denaturation of the purified proteins and is thus not applicable to proteins that do not spontaneously refold.

高效液相層析法
高性能液相層析或高壓液相層析法, 是層析法是一種類形, 施加高的壓力來驅動的溶質快些通過層析層析分離管. 這意味著, 其擴散被限制了, 分離的解析度被提高了.
最常見的類形是 "逆相HPLC"(高壓液相層析法), 層析分離管是疏水性的. 蛋白質是經由漸增有機溶劑量(如乙腈)的溶劑比例梯度. 蛋白質是根據其疏水性程度的差異被沖滌出來.
經HPLC純化後的蛋白質是在只包含揮發性化合物溶液中, 可以很容易地被冷凍乾燥.
高效液相層析(HPLC) 分離經常導致純化蛋白質的變性, 因此不適用於不會自發地重新折疊的蛋白質.

Concentration of the purified protein 純化的蛋白質的濃縮
A selectively permeable membrane can be mounted in a centrifuge tube. The buffer is forced through the membrane by centrifugation, leaving the protein in the upper chamber. At the end of a protein purification, the protein often has to be concentrated. Different methods exist.

一個選擇性滲透膜可被安裝在一個離心管中. 緩衝溶液經由離心作用, 被強制通過濾膜, 在膜上空間留下了蛋白質. 在蛋白質純化的結束步驟, 蛋白質通常必被濃縮. 存在不同的方法.

Lyophilization 冷凍乾燥
If the solution doesn't contain any other soluble component than the protein in question the protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile components, leaving the proteins behind.

如果溶液不包含任何其他可溶成分, 則比蛋白可被冷凍乾燥. 這通常是在跑過高效液相層析之後進行的. 這只是簡單地將所有揮發性成分移除, 將蛋白質留下.

Ultrafiltration
超過濾 Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining the protein. The solution is forced against the membrane by mechanical pump, gas pressure, or centrifugation.

超過濾是使用選擇性滲透膜, 以濃蘇縮蛋白質溶液. 超過濾的功能是讓水和小分子通過, 同時保留了蛋白質. 溶液是以由機械幫蒲, 氣體壓力, 或離心分離等方式對濾膜施力.

Analytical 分析(目的的)
Denaturing-Condition Electrophoresis 變性條件電泳
Gel electrophoresis is a common laboratory technique that can be used both as preparative and analytical method.
The principle of electrophoresis relies on the movement of a charged ion in an electric field.
In practice, the proteins are denatured in a solution containing a detergent (SDS). In these conditions, the proteins are unfolded and coated with negatively charged detergent molecules.
The proteins in SDS-PAGE are separated on the sole basis of their size. In analytical methods, the protein migrate as bands based on size. Each band can be detected using stains such as Coomassie blue dye or silver stain.
Preparative methods to purify large amounts of protein, require the extraction of the protein from the electrophoretic gel. This extraction may involve excision of the gel containing a band, or eluting the band directly off the gel as it runs off the end of the gel.
In the context of a purification strategy, denaturing condition electrophoresis provides an improved resolution over size exclusion chromatography, but does not scale to large quantity of proteins in a sample as well as the late chromatography columns.

凝膠電泳是一種常見的實驗室技術,可用於作為製備和分析方法.
電泳的原理依賴於在電場中的電荷的離子的移動. 在實務上, 蛋白質是在含有清潔劑(SDS)的溶液中被變性. 在這些條件下,  蛋白質是被展開並塗有帶負電的清潔劑分子.
蛋白質在SDS-PAGE分離法裏的唯一依據是尺吋的大小.
在分析方法中,蛋白質的基於其尺吋的大小的有不同條帶的遷移. 
每個條帶可以用考馬斯亮藍染色或銀染色的斑點被檢測.
大量的蛋白質純化的製備方法中, 需要從電泳凝膠上取出蛋白質. 此提取過程可能涉及含的條帶的凝膠部份的切除, 或在其跑到凝膠尾端時, 直家接將條帶沖滌下來. 
在純化策略的關聯中, 變性條件電泳提供在解析度超過斥慮層析法改進,
但其尺度並不能擴展到像後來的管柱層析樣品中的大量蛋的白質.

Non-Denaturing-Condition Electrophoresis 非變性條件電泳

An important non-denaturing electrophoretic procedure for isolating bioactive metalloproteins in complex protein mixtures is termed 'quantitative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE).

一個重要的非變性電泳程序, 用以分離複雜的蛋白質混合物中的生物活性的金屬蛋白質, 被稱為“定量的固有連續聚丙烯酰胺凝膠電泳(QPNC-PAGE).

References[edit] 參考
^ "The Nobel Prize in Chemistry 1946". Retrieved 2011-09-19.
^ Ehle H, Horn A (1990). "Immunoaffinity chromatography of enzymes". Bioseparation 1 (2): 97–110. PMID 1368167.
^ Regnier FE (October 1983). "High-performance liquid chromatography of biopolymers". Science 222 (4621): 245–52. doi:10.1126/science.6353575. PMID 6353575.

External links 外部鏈接
Protein purification in one day
Protein purification facility
Slope Spectroscopy

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台中 伴手禮 文化節 禮物 母親節 端午節 高美濕地 佳節倍思親,好禮表孝心, 父母的健康,就是子女的孝心. 佳節好禮, WWW.chromnet.net 穩達F3靈芝多醣體在使用中可持續保有可視圖文的肥皂 (Soaps with Sustained Visible Characters and Graphics while applying) 一.特點, 二.可客製化設計三.訂購方式特點: (專利申請中:101150476, 102105651)  1.含有金玉良言及圖案 2.一直洗,一直洗,仍可看得到圖文 3.可多層組成一組詩詞短句本項發明的目的在於提供各種內含有圖案或文字的肥皂, 此圖案或文字具有深度方向的厚度, 以使得在肥皂的在使用過程中, 仍可持續保有可以被看到的圖案或文字. The purpose of this invention is to provide soaps with characters and graphics included. The included characters and graphics have their depths in the thickness direction to sustain their visibilities while applying the soaps. 現有的各種肥皂所內含的圖案或文字, 主要都以表面淺層凹版或凸版方式製作, 在短暫的使用後, 肥皂表面的圖案或文字即被消耗而看不見了, 因而未能在其使用過程中持續保有可以被看到的圖案或文字. 本項發明的目的在於提供各種內含有圖案或文字的肥皂, 此圖案或文字具有深度方向的厚度, 以使得在肥皂的在使用過程中, 仍可持續保有可以被看到的圖案或文字.本系統發明的重點在於其應用方式, 組構型態及製作, 所用零組件能達到所須功能即可. 利用各種常見或特定的肥皂的材料及色料, 製作含有圖案或文字的肥皂,此圖案或文字具有深度方向的厚度. 可客製化設計: 我們可以依據您們的指定,設計所須要的"深度圖文耐用皂", 包括: 皂體尺寸,圖文內容,皂體顏色,圖文顏色,香精成份,單一皂體包裝材質及印刷內容,多皂體禮盒構造材質及印刷內容等... "深度圖文耐用皂" 是可以重疊多層,形成一個含有多層文字圖案的耐用皂,其特點是可在一個多層文字圖案耐用皂裏表達出一篇簡短的古詩,現代詩,對聯或短句等,是我們的專利主要訴求之一.  另我們也可有涵蓋到固體芳香劑及馬桶小便斗除臭劑的應用上,使得芳香劑及馬桶小便斗除臭劑上可以有不同顏色的文字或線條圖案,如"早安"或"請對準","花朵"或"蒼蠅"(圖案),深度都是與固體芳香劑及馬桶小便斗除臭劑的厚度一樣,在用完以前就可以一直被看到,這也是既實用有創新的應用. 客製化的設計, 可以增進客戶的在創新服務層面的評價, 使得產品及服務內容更具有吸引力. 訂購方式: 請至各子項目查看各現有產品訂價,確定所需品項,香精種類及數量之後,請直接以EMAIL連繫,經我們確定可交件日期後,即可付款訂購. 常見香精有:香茅,艾草,薰衣草,樟樹,茶樹,玫瑰麝香,甜菊,月桂,... 運費及運送方式:一次購滿500元免運費,500元以下酌收50-80元運費.以郵局包裹方式運送.  匯款資訊:台中市清水區農會 活期存款帳號名稱:林坤益 (+) 太陽系分析基礎開發中心, 帳號: 45000100033625 通匯代碼: 9544505 NOTE:1.香精種類很多,部份香精對特定體質具有刺激性或過敏性,一般建議先少量短期使用,以確保適用. NOTE:2.不同成份的香精成本差距相當大(5-20倍),在您確認訂購時,會適度酌收高單價香精的成本費用. NOTE:3.不加香精也是種很好的選項,完全不用顧慮刺激性或過敏性問題,但使用時會有甘油皂基的原本不香的味道.能夠體認習慣了也是很好的選項. NOTE4:客製化部份, 煩均以email連繫及確認需求及可交件日期,     良言一句三冬暖(星雲--遠見雜誌)  語言是思想和觀念的表象, 用來聯繫人心的般若風光. 若能時時慎之於口, 與他人共享, 陽光,花朵.淨水般的話語, 人生必然豐美... (http://www.gvm.com.tw/Boardcontent_4574.html) 多年以前,曾經在一篇文章裡,讀到這麼一句話:「語言,要像陽光、花朵、淨水。」當時深深感到十分受用,於是謹記心田,時刻反省,隨著年歲的增長,益發覺得其中意味深長。 http://www.sfes.tc.edu.tw/index.asp(上楓國小好話實踐) http://www.sfes.tc.edu.tw/index.asp(上楓國小唐詩河洛語線上教學)       節氣指二十四時節和氣候,是中國古代訂立的一種用來指導農事的補充曆法,... http://zh.wikipedia.org/wiki/节气二十四節氣(農委會)http://www.coa.gov.tw/view.php?catid=284 今日星象、行星動向、節氣 http://web2.nmns.edu.tw/constellation/home.php AEEA 天文教育資訊網 http://aeea.nmns.edu.tw/ 太陽系的前世今生, 影像提供:NASA/ESA,陳輝樺(NMNS) http://aeea.nmns.edu.tw/index1.html   大地藏無盡, 勤勞資有生, 念哉斯意厚,努力事春耕. 一.特點,二.可客製化設計,三.訂購方式   大地藏無盡  念哉斯意厚 勤勞資有生 努力事春耕298元/組(320g) 誠歡迎有天文學,民俗學及相關的教育及創新構想的原創者及推廣者,與我們密切合作 !    氨基酸分子模型系列(19種常見氨基酸) 由丙氨酸的基礎結構可研衍生出各種常見氨基酸分子結構一.特點,二.可客製化設計,三.訂購方式   分子球 89元/個(80g)    丙安酸(Alanine)  丙氨酸 369元/個(330g) 麩氨酸(圓柱) 469元/個(430g) 誠歡迎有科學教育創新構想的原創者及推廣者,與我們密切合作 ! RainbowDash 及UNLIGHT 是最經點的動漫符號之一網路搜尋即有豐富的資訊. Ranbow Dash 動畫My Little Pony:Friendship is Magic 之主角群之一 Cutie mark的圖案為打著彩虹閃電的雲在故事中代表的精神為"忠誠" UNLIGHT FACEBOOK網頁遊戲以故事為中心的對戰型卡牌養成遊戲玩家人數突破30萬人   好香皂結合好男人及撿肥皂的梗,並將其實體化梗的詳細資料有請 網路搜尋   誠歡迎有文化及新構想的原創者及推廣者,與我們密切合作. 先民從大陸移居台灣,渡過黑水溝,也把馬祖信仰帶來台灣。媽祖信仰起源於西元10世紀的宋朝,幾乎每個台灣人都耳熟能詳福建莆田湄洲林默娘傳說,為了指引出海的父兄返航而犧牲自己,後來成為漁民信仰的「航海女神」。 一.特點,二.可客製化設計,三.訂購方式   保庇 89元/個(80g)     恭賀新喜_1 79元/個(30g)      金鎖片 79元/個(80g) 元寶錢幣 79元/個(80g)   風調雨順 99元/個(80g) 國泰民安_2 99元/個(80g) 富貴吉祥_1 99元/個(80g) 年年有餘_1 79元/個(80g) 年年有餘_3 99元/個(80g) 誠歡迎有宗教學,民俗學及相關的教育及創新構想的原創者及推廣者,與我們密切合作 ! 牛牽到北京 還是牛,.. 一.特點,二.可客製化設計,三.訂購方式    牛牽到北京   牛牽到北京還是牛 188元/組(160g) 還是牛 台灣諺語_動物篇 http://163.21.2.41/t128/chinesepassport/animals.htm 動物之台灣諺語 www.taiwan123.com.tw/local/a_index.htm 誠歡迎有趣味文學相關的教育及創新構想的原創者及推廣者,與我們密切合作 ! 沒辦法再更傳統了特出的文化產品, 如沐春風粿模經典(雕:餅印.粿印.糕印.糖印) 粿模_財子壽中草藥種苗很專業的中草藥種苗來源, 值得珍藏綬草沒辦法再更先進了用最先進的技術, 提供最優化的生活營養保健美肌保養居家潔身居家潔身深度圖文耐用手工皂唯一可以讓我們的日常生活充滿文學品味的手工皂, 具有無可替代的專利特點發現金玉良言系列良言一句三冬暖媽祖(風調雨順, 國泰民安, 保庇每一個手工皂都有一句媽祖的提示良言, 直到用完前都還能看得見節氣(驚執:大地藏無盡, 勤勞資有生, 念哉斯意厚, 努力事春耕清明節母親節端午節畢業紀念四書, 五經經典文學系列, 詩, 詞, 曲勵志追思永懷親恩祝賀五世其昌情意與關懷 LOVE, WISH, 平安文具禮頻贈品不只是高CP值,還特別具有創新及創意科教禮贈用品結合科學, 教育, 文創, 既有趣又有豐富的科學人文內涵天文科學星座觀測, 觀測工具發現生命的起源及意義考古人類學探索, 學習, 驚奇, 有趣 ... 追思(城鄉差距與世代正義) 記憶伴隨著歲月, 從鄉村到城市, 一切是否留下寶貴的智慧, 或是只是無限的追思 ! 小時候, 母親曾說:"如果我們這邊有一條較直的大路直接到清水鎮上就好了" 這事實上也是我們鄉下人共同的期望與夢想. 記得每年農曆10月13前後, 母親難得年回甲南娘家一次 , 我們總是既期待又害羞. 回甲南娘家其實不遠, 走路只要約50-60分鐘 , 不過幾十年來一直也沒有公車... 有時後總是在想, 這人類的社會到底是怎麼回事 ? 人口總是往都市集中, 留在鄉下卻是一些無法被提起的記憶 ? 因此, 容我們有一點能力與機會, 來訴說我們的所見所知與所感 , 也希望這一切是留下來的不只是無限的追思.