JPS6195241A - Portionwise sampling carrier-free electrophoretic method - Google Patents
Portionwise sampling carrier-free electrophoretic methodInfo
- Publication number
- JPS6195241A JPS6195241A JP59216286A JP21628684A JPS6195241A JP S6195241 A JPS6195241 A JP S6195241A JP 59216286 A JP59216286 A JP 59216286A JP 21628684 A JP21628684 A JP 21628684A JP S6195241 A JPS6195241 A JP S6195241A
- Authority
- JP
- Japan
- Prior art keywords
- solvent
- electrolyte
- carrier
- free
- electrophoresis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
- B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
Abstract
Description
【発明の詳細な説明】
〔発明の利用分野〕
本発明は、遺伝子工学、細胞工学の分野におけるタンパ
ク質、核酸、細胞などの電解質を分離分取するための電
気泳動法にかかわり、特に、大量に分離分取するに好適
な分取用無担体電気泳動法に関するものである。[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to an electrophoresis method for separating and fractionating electrolytes of proteins, nucleic acids, cells, etc. in the fields of genetic engineering and cell engineering, and in particular, The present invention relates to a preparative carrier-free electrophoresis method suitable for separation and fractionation.
分取用電気泳動法については、従来、マックスブランク
生化学研究所のＰｒｏｆ、 Ｄｒ、 Ｈａｎｎｉｇによ
る“Ｎｅｗ ａｓｒｐｅｃｔｓ ｉｎ ｐｒｅｐａｒａ
ｔｉｖｅ ａｎｄ ａｎａｌｙｔｉｃａｌｃｏｎｔｉｎ
ｕｏｕｓ ｆｒｅｅ−ｆｌｏｗ ｃｅｌｌ ａｌｅｃｔ
ｒｏｐｈｏｒｅｓｉｓ”。Regarding the preparative electrophoresis method, there is a conventional method such as "New asrpects in prepara" by Prof. Dr. Hannig of Max Blank Institute for Biochemistry.
tive and analytical content
uous free-flow cell align
rophoresis”.
Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ、 ３ 、２３５〜２
４３（１９８２）と題する文献において、また特開昭５
７−２４８５１号公報において、論じられている。 Ｈ
ａｎｎｉｇの方法では、分離されるべき電解質試料は、
電場を横切って流下する泳動槽内の分離用溶媒の中に細
い注入孔から連続的に注入される。各電解質は種々に帯
電しているため、各電解質の粒子の流路は、溶媒の流速
と電解質の電荷量との兼ね合いで、溶媒の流下方向に対
して当該電解質の電荷量に特有の角度をとる。Electrophoresis, 3, 235-2
43 (1982), and also in JP-A No. 5
Discussed in Publication No. 7-24851. H
In the annig method, the electrolyte sample to be separated is
It is continuously injected through a narrow injection hole into the separation solvent in the electrophoresis tank that flows down across the electric field. Since each electrolyte is charged differently, the flow path of particles of each electrolyte has an angle specific to the amount of charge of the electrolyte with respect to the direction of flow of the solvent, depending on the flow rate of the solvent and the amount of charge of the electrolyte. Take.
その結果、電荷量の等しい電解質はそれぞれ帯状になっ
て流下し１分画して連続して分取される。As a result, the electrolytes having the same amount of charge flow down in the form of bands and are successively separated into one fraction.
上記の方法は、電解質試料を連続的に添加し。The above method involves adding the electrolyte sample continuously.
分離分取することができるため、遺伝子工学や細胞工学
の企業化に必要なタンパク質、核酸、細胞の大量分離精
製に有効な提案である。しかし、この方法では、電場を
横切って流下する溶媒の流下速度を泳動槽内で均一にす
る必要があるが、溶媒に電流が流れてジュール熱が発生
するので、溶媒内に対流が発生し、このため、溶媒の流
下速度を均一にすることは難しく、高い分離能や再現性
が得られないという短所がある。また、この方法によっ
て分離能と再現性をある程度維持するためには、溶媒の
流速と温度を±０．２％以内に維持する必要があるが、
この条件の下では、小型の装置（例えば、長さ１３０ｍ
ｍ、幅３０ｍｍ、厚さ０．２間程度のもの）しか実用化
できず、しかも分離能はさほど良くないという問題があ
る。Since it can be separated and fractionated, it is an effective proposal for separating and purifying large quantities of proteins, nucleic acids, and cells, which is necessary for the commercialization of genetic engineering and cell engineering. However, in this method, it is necessary to make the velocity of the solvent flowing down across the electric field uniform within the electrophoresis tank, but as a current flows through the solvent and generates Joule heat, convection occurs within the solvent. For this reason, it is difficult to make the flow rate of the solvent uniform, and there is a disadvantage that high separation power and reproducibility cannot be obtained. In addition, in order to maintain a certain degree of separation power and reproducibility with this method, it is necessary to maintain the solvent flow rate and temperature within ±0.2%.
Under these conditions, small equipment (e.g. 130 m long)
There is a problem in that only those with a width of about 30 mm, a width of about 30 mm, and a thickness of about 0.2 mm can be put into practical use, and the resolution is not very good.
本発明の目的は、遺伝子工学や細胞工学の企業化に必要
なタンパク質、核酸、細胞などの電解質を高分離能で大
量に分離分取することができる分取用無担体電気泳動法
を提供することにある。The purpose of the present invention is to provide a preparative carrier-free electrophoresis method that can separate and fractionate electrolytes from proteins, nucleic acids, cells, etc., in large quantities with high resolution, which are necessary for the commercialization of genetic engineering and cell engineering. There is a particular thing.
本発明は、従来の方法の難点が溶媒の流速と温度のコン
トロールにあることに鑑み、溶媒の流速や温度を高精度
にコントロールすることなしに高い分離能と再現性が得
られるように留意してなされたもので、その主要点は、
（イ）電気泳動中、溶媒の水素イオン指数ｐＨを変化さ
せることにより、それぞれのｐＨに対して保持されてい
る電解質のうち一定の電荷をもつもののみを分離回収し
、電解質の分離分取を行う。In view of the difficulty of conventional methods in controlling the flow rate and temperature of the solvent, the present invention takes care to obtain high resolution and reproducibility without precisely controlling the flow rate and temperature of the solvent. The main points are: (a) During electrophoresis, by changing the hydrogen ion index pH of the solvent, only those electrolytes with a constant charge are retained for each pH. The electrolyte is separated and collected, and the electrolyte is separated and fractionated.
（ロ）電気的吸引力あるいは電場の中で溶媒を一定方向
に強制的に対流させることにより、回収すべき電解質を
一定部位に保持し、その電解質を回収する。(b) By forcing the solvent to convect in a certain direction in an electric attraction force or an electric field, the electrolyte to be recovered is held in a certain position and the electrolyte is recovered.
の２点である。There are two points.
自由溶媒中に保持された電解質が電気泳動する場合、電
解質の粒子は、あるものは電極へ、あるものは−極へ移
動するが、いずれの極へ移動するかは当該電解質の等電
点と溶媒の水素イオン指数ｐＨに依存する。従って、溶
媒のｐＨを適当に選べば、一定電荷を有する電解質のみ
の粒子が電極（または−極）へ、他のすべての電解質の
粒子は一極（または電極）へ移動するようにさせること
ができる。そこで、前者の電解質を回収し、ついでｐＨ
を変化させて同様な操作を繰り返し、電解質を一つずつ
回収していけば、？８媒に保持されている電解質を分離
分取することができる。これが第１の要点である。When an electrolyte held in a free solvent undergoes electrophoresis, some electrolyte particles move to the electrode and others to the - pole, but which pole they move to depends on the isoelectric point of the electrolyte. The hydrogen ion index of the solvent depends on the pH. Therefore, if the pH of the solvent is appropriately selected, particles of only the electrolyte with a certain charge can be made to migrate to the electrode (or - pole), and all other electrolyte particles can be made to migrate to the single pole (or electrode). can. Therefore, the former electrolyte was recovered, and then the pH
What if we repeat the same operation with different values and collect the electrolytes one by one? The electrolyte held in the 8 medium can be separated and fractionated. This is the first point.
また、上記のように、本発明では電解質を一方の電極側
に集めて回収していくが、溶媒を一定方向に強制的に対
流させ、電解質を一定部位に保持すれば、その電Ｎ質を
容易に回収できる。これが第２の要点である。In addition, as mentioned above, in the present invention, the electrolyte is collected by collecting it on one electrode side, but if the solvent is forced to convect in a certain direction and the electrolyte is held in a certain position, the electrolyte can be collected. Can be easily recovered. This is the second point.
以下、本発明の一実施例を第１図により説明する。図に
おいて、ガラス製の容器１の内部には一定のｐＨの溶媒
が充填されており、この溶媒はイオン透過性の膜４によ
って電気泳動槽２内とｐＨ変換槽３内とに分離されてい
る。５は中電極、６は一電極で、両電極はイオン透過性
の膜を介して電気泳動槽２内の溶媒に結合している６電
気泳動槽２の内部の生電極５側には、溶媒を対流させる
ため加熱器７が取り付けである。また、８はサンプル添
加孔、９はサンプル回収孔である。試料には。An embodiment of the present invention will be described below with reference to FIG. In the figure, a glass container 1 is filled with a solvent having a constant pH, and this solvent is separated into an electrophoresis tank 2 and a pH conversion tank 3 by an ion-permeable membrane 4. . 5 is a middle electrode, 6 is one electrode, and both electrodes are bonded to the solvent in the electrophoresis tank 2 through an ion-permeable membrane. 6 The live electrode 5 side inside the electrophoresis tank 2 has a solvent A heater 7 is attached to cause convection. Further, 8 is a sample addition hole, and 9 is a sample collection hole. For the sample.
卵白リゾチーム 等電点 １１〜１１．４ミオグロビ
ン 等電点 ８．１〜８．２ダイズトリプシンイン
ヒビター
等電点 ４．３〜４．６
の３種類のタンパク質を使用した。Three types of proteins were used: egg white lysozyme with an isoelectric point of 11-11.4, myoglobin with an isoelectric point of 8.1-8.2, and soybean trypsin inhibitor with an isoelectric point of 4.3-4.6.
次に、動作について説明する。まず１分離の第１段階と
して、容器１の内部にｐＨ８，９の溶媒を満たし、電極
５，６間に電圧をかけ、上記３種のりンパク質のｐＨ８
，９の溶液をサンプル添加孔８から注入する。溶媒中に
注入されたタンパク質は、溶媒のｐＨが８．９であるた
め、３種のうち、正に荷電された等電点１１〜１１．４
の卵白リゾチームの粒子のみが一電極側へ、負に荷電さ
れた他の２種のタンパク質の粒子は生型極側へ移動しよ
うとする。一方、溶媒は加熱器７により加熱され、太矢
印で示すような対流を生じている。その結果、卵白リゾ
チームは矢印Ａで示すように流れ、下の方に集まり、他
方、ミオグロビン、ダイズトリプシンインヒビターは矢
印Ｂで示すように流れる。この時点で、サンプル回収孔
９からタンパク質を含む溶液を回収することにより、卵
白リゾチームのみを分＃ｉ回収する。卵白リゾチームが
すべて回収されたところで、第２段階として、ｐＨ変換
椿３内部の１ 溶媒をｐＨ６・Ｏに変更し・電
気泳動槽２内の溶媒をｐＨ６，０に変え、同様な操作を
行って１等電点８．１〜８．２のミオグロビンのみをサ
ンプル回収孔９から分離回収する。最後に、電気泳動槽
２内の溶媒をｐＨ３に調整すると、ダイズトリプシンイ
ンヒビターが回収される０以上のような手順によって、
試料の３種のタンパク質を分離分取することかできる。Next, the operation will be explained. First, in the first step of 1 separation, the inside of the container 1 is filled with a solvent with a pH of 8 and 9, and a voltage is applied between the electrodes 5 and 6, so that the pH of the three types of proteins mentioned above is 8 and 9.
, 9 are injected through the sample addition hole 8. Since the pH of the solvent is 8.9, the protein injected into the solvent has a positively charged isoelectric point of 11 to 11.4 among the three species.
Only the egg white lysozyme particles try to move toward one electrode, while the other two negatively charged protein particles try to move toward the living electrode. On the other hand, the solvent is heated by the heater 7, causing convection as shown by thick arrows. As a result, egg white lysozyme flows as shown by arrow A and gathers at the bottom, while myoglobin and soybean trypsin inhibitor flow as shown by arrow B. At this point, only the egg white lysozyme is collected by #i by collecting the solution containing the protein from the sample collection hole 9. When all of the egg white lysozyme was collected, in the second step, the 1 solvent inside the pH converting Tsubaki 3 was changed to pH 6.0, and the solvent in the electrophoresis tank 2 was changed to pH 6.0, and the same operation was performed. Only myoglobin with a first isoelectric point of 8.1 to 8.2 is separated and collected from the sample collection hole 9. Finally, when the solvent in the electrophoresis tank 2 is adjusted to pH 3, the soybean trypsin inhibitor is recovered by the procedure described above.
It is possible to separate and fractionate three types of proteins in a sample.
第１図に示した実施例では、溶媒を強制的に対流させる
手段として加熱器７を使用しているが、この手段の他の
例を第２図、第３図に示す、第２図では、電気泳動槽内
の溶媒を対流させるのに、冷却水管１１と温水管１２と
を使用し、槽内の溶媒に＋電極側と一電極側とで温度差
を与え、対流をさせる。また、第３図では、電気泳動槽
内の溶媒を対流させるのに、かく弾機２１を使用してい
る。In the embodiment shown in FIG. 1, a heater 7 is used as a means for forcibly convecting the solvent, but other examples of this means are shown in FIGS. To cause convection of the solvent in the electrophoresis tank, a cooling water pipe 11 and a hot water pipe 12 are used, and a temperature difference is given to the solvent in the tank between the + electrode side and the one electrode side to cause convection. Further, in FIG. 3, an agitator 21 is used to cause convection of the solvent in the electrophoresis tank.
第４図と第５図とは、第１図により説明した実施例の変
形を示したものである。第４図では、電気分解による電
極近傍のｐＨ変化を防止するため、ポンプ３１によって
生電極５および一電極６周辺の溶媒を少量ずつ入れ替え
ている１図中、３２．３３はｐＨ変換槽と電極周辺の溶
媒をつなぐ管である。4 and 5 show a modification of the embodiment described with reference to FIG. 1. FIG. In Figure 4, in order to prevent pH changes near the electrodes due to electrolysis, the solvent around the live electrode 5 and one electrode 6 is replaced little by little by the pump 31. In Figure 1, 32.33 indicates the pH conversion tank and the electrode. This is a tube that connects the surrounding solvent.
また、第５図は、電気泳動槽内における電解質の
ｌ゛分離より確実にするため、槽の中央部に遮へ
い板４１を設けである。Figure 5 also shows the electrolyte in the electrophoresis tank.
1) To ensure more reliable separation, a shielding plate 41 is provided in the center of the tank.
第６図は本発明の他の実施例を示したものである。図に
おいて、５Ｉはガラス製の容器、５２は電気泳動槽、５
３はｐＨ変換槽、５４はイオン透過性の膜。FIG. 6 shows another embodiment of the invention. In the figure, 5I is a glass container, 52 is an electrophoresis tank, and 5
3 is a pH conversion tank, and 54 is an ion-permeable membrane.
５５は生電極％５６は一電極、５８はサンプル添加孔。55 is a raw electrode, 56 is one electrode, and 58 is a sample addition hole.
５９はサンプル回収孔である。本実施例では、電気泳動
槽５２内の溶媒に対流を起こさせる代りに、電気的吸引
力と拡散板６０．６１により、電解質の不規則な拡散を
防いでいる。本実施例においても、第１図により説明し
た実施例の場合と同様に、溶媒のｐＨを段階的に下げる
ことにより、生型極側に保持されている電解質が、等電
点の高い順にサンプル回収孔５９から回収され、分離さ
れる。59 is a sample collection hole. In this embodiment, instead of causing convection in the solvent in the electrophoresis tank 52, irregular diffusion of the electrolyte is prevented by electric attraction and diffusion plates 60, 61. In this example as well, as in the example explained with reference to FIG. It is collected through the collection hole 59 and separated.
本発明によれば、分取用無担体電気泳動法において、電
気泳動中に発生するジュール熱に起因する分離能の低下
が起こらないので、タンパク質、核酸、細胞などの電解
質を高分８「能で大量に分離分取することが可能となる
。According to the present invention, in preparative carrier-free electrophoresis, there is no reduction in separation ability due to Joule heat generated during electrophoresis, so electrolytes such as proteins, nucleic acids, cells, etc. It becomes possible to separate and fractionate large quantities.
第１図は本発明による分取用無担体電気泳動法の一実施
例に使用する装置の説明図、第２図と第３図は該実施例
で用いる溶媒を対流させる手段の他の例を示す説明図、
第４図と第５図は第１図の実施例の変形例を示す説明図
、第６図は本発明の他の実施例に使用する装置の説明図
である。
符号の説明
１・・・ガラス製の容器 ２・・・電気泳動槽３・・
・ｐＨ変換槽 ４・・・イオン透過性の膜５・・
・生電極 ６・・・−電極７・・・加熱器
８・・・サンプル添加孔９・・・サンプル回
収孔 １１・・・冷却水管１２・・・温水管
２１・・・かく弾機３１・・・ポンプ
３２）３３・・・ｐＨ変換槽と電極周辺の溶媒をつなぐ
管４１・・・遮蔽板 ５２・・・電気泳動槽
５３・・・ｐＨ変換槽 ５４・・・イオン透過性
の膜５５・・・生電極 ５６・・・−電極５
８・・・サンプル添加孔 ５９・・・サンプル回収孔
６０、６１・・・拡散板FIG. 1 is an explanatory diagram of an apparatus used in an embodiment of the preparative carrier-free electrophoresis method according to the present invention, and FIGS. 2 and 3 show other examples of means for convection of a solvent used in the embodiment. An explanatory diagram showing,
4 and 5 are explanatory diagrams showing a modification of the embodiment of FIG. 1, and FIG. 6 is an explanatory diagram of an apparatus used in another embodiment of the present invention. Explanation of symbols 1...Glass container 2...Electrophoresis tank 3...
・pH conversion tank 4...Ion permeable membrane 5...
・Live electrode 6...-electrode 7...heater
8...Sample addition hole 9...Sample collection hole 11...Cooling water pipe 12...Hot water pipe
21... Crater 31... Pump 32) 33... Tube connecting the pH conversion tank and the solvent around the electrode 41... Shielding plate 52... Electrophoresis tank 53... pH conversion tank 54 ... Ion-permeable membrane 55 ... Live electrode 56 ... - Electrode 5
8... Sample addition hole 59... Sample collection hole 60, 61... Diffusion plate
Claims (2)
、該溶媒に保持された電解質を電気泳動させて該電解質
を分離分取する分取用無担体電気泳動法であって、溶媒
の水素イオン指数ｐＨを選択して、一定電荷を有する電
解質のみの粒子を一方の電極へ移動させ、該電極側に移
動した電解質を回収し、以下水素イオン指数ｐＨを変え
て同様な操作を繰り返し、電気泳動中に溶媒の水素イオ
ン指数ｐＨを段階的に変化させて電解質を一定電荷を有
するもの一つずつ回収することにより該溶媒に保持され
た電解質を分離分取することを特徴とする分取用無担体
電気泳動法。(1) A preparative carrier-free electrophoresis method in which a voltage is applied between a pair of electrodes provided in a free solvent, and the electrolyte retained in the solvent is electrophoresed to separate and fractionate the electrolyte, comprising: Select the hydrogen ion index pH of the solvent, move particles of only electrolyte with a constant charge to one electrode, collect the electrolyte that has moved to the electrode side, and repeat the same operation by changing the hydrogen ion index pH. The method is characterized in that the electrolyte retained in the solvent is separated and fractionated by repeatedly changing the hydrogen ion index pH of the solvent stepwise during electrophoresis and recovering electrolytes having a constant charge one by one. Preparative carrier-free electrophoresis method.
泳動法において、電気泳動中溶媒を一定方向に強制的に
対流させる手段を設け、回収すべき電解質を溶媒中の一
定部位に保持するようにしたことを特徴とする分取用無
担体電気泳動法。(2) In the preparative carrier-free electrophoresis method described in claim 1, a means for forcibly convecting the solvent in a certain direction during electrophoresis is provided, and the electrolyte to be recovered is transferred to a certain part in the solvent. A preparative carrier-free electrophoresis method characterized in that the carrier-free electrophoresis method retains
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59216286A JPS6195241A (en) | 1984-10-17 | 1984-10-17 | Portionwise sampling carrier-free electrophoretic method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59216286A JPS6195241A (en) | 1984-10-17 | 1984-10-17 | Portionwise sampling carrier-free electrophoretic method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6195241A true JPS6195241A (en) | 1986-05-14 |
Family
ID=16686152
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59216286A Pending JPS6195241A (en) | 1984-10-17 | 1984-10-17 | Portionwise sampling carrier-free electrophoretic method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6195241A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7316771B2 (en) | 2000-09-21 | 2008-01-08 | Becton, Dickinson And Company | Medium for analytic and preparative electrophoresis |
| US7399394B2 (en) | 2000-12-18 | 2008-07-15 | Becton, Dickinson And Company | Electrophoresis device, electrophoresis method using an electrophoresis device and use of the electrophoresis device |
| US7491304B2 (en) | 2000-12-18 | 2009-02-17 | Becton, Dickinson And Company | Carrierless electrophoresis process and electrophoresis device for carrying out this process |
| US8721861B2 (en) | 2005-04-29 | 2014-05-13 | Becton, Dickinson And Company | Method for electrophoresis involving parallel and simultaneous separation |
-
1984
- 1984-10-17 JP JP59216286A patent/JPS6195241A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7316771B2 (en) | 2000-09-21 | 2008-01-08 | Becton, Dickinson And Company | Medium for analytic and preparative electrophoresis |
| US7399394B2 (en) | 2000-12-18 | 2008-07-15 | Becton, Dickinson And Company | Electrophoresis device, electrophoresis method using an electrophoresis device and use of the electrophoresis device |
| US7491304B2 (en) | 2000-12-18 | 2009-02-17 | Becton, Dickinson And Company | Carrierless electrophoresis process and electrophoresis device for carrying out this process |
| US8721861B2 (en) | 2005-04-29 | 2014-05-13 | Becton, Dickinson And Company | Method for electrophoresis involving parallel and simultaneous separation |
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