JPS58105052A - Simple method of two-dimentional electrophoresis - Google Patents

Abstract

PURPOSE:To enable the substantial simplification of an operation of separating electrophoresis, by using the same plane-shaped gel for one-dimentional and two- dimentional electro-phoresis without distinguishing gels used for these two stages of electrophoresis from each other, and thereby enabling the handling of the gel together with a hard substrate. CONSTITUTION:One surface of a glass plate 1 is processed by methacryl oxypropyltrimethoxysilane, and thereby a plane gel 2 having the concentration gradient of acrylamide of 4-20% and containing potassium chloride is prepared. A cut 3 is made in the low-concentration part of the gel and a serum and amphorine, which is an amphoteric electrolyte, are given in it. Filter paper sheets 4 and 5 for liquid junction are connected to a negative electrode tank 9 and a positive electrode tank 10, and serum protein is separated according to the difference in an isoelectric point. A filter paper sheet impregnated with a buffer (A liquid) containing trishydroxymethyl-aminomethane and glycine is put on, the A liquid is made to permeate the plane gel 2, the filter paper sheet is removed, filter paper sheets 7 and 8 for liquid junction are connected to a positive electrode tank 11 and a negative electrode tank 12, and the serum protein is separated according to the difference in molecular weight.

Description

【発明の詳細な説明】 本発明に臨床検査を目的とした血液などの体液の分析法
に係り、％に、体液中に含ま扛る諸種蛋白質の多成分同
時分析に好適な二次元電気泳動分析法に関するものであ
る。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for analyzing body fluids such as blood for the purpose of clinical testing, and includes two-dimensional electrophoretic analysis suitable for simultaneous multi-component analysis of various proteins contained in body fluids. It is about law.

従来、血液蛋白質の多成分同時分析法として用いらｔて
いるポリアクリルアミドケルを支持体とする二次元電気
泳動分析法（詳＃ｌｒｉ例えは［蛋白質・核酸−酵素」
、共立出版、１９７８年９月号２１１頁参照）において
は、１づ、両性電解實を含む細長い円筒状のゲルをカラ
ス管内に作成し、ケルの両端に電圧をかけ、ケルの一端
に添加した血液試料中の成分蛋白質をその等電点差によ
り分離する（一次元目の分離）。次に、別に、２枚のガ
ラス板の間に作成しておいたアクリルアミドのＳ度勾配
を有する平板状のケルの低濃度端に、一次元目の分離を
終了した円筒状のケルをカラス管から押し出して乗せ、
改めて平板ケルの濃度勾配方向に通電して二次丸目の分
子Ｉ差による分１１１ｔを行々う。次に、各分離蛋白質
を定量するために、平板ケルをＩ″ｉざんでいるガラス
板２枚會にすし、例えは、コーマツシーブルーなどの染
料溶液にゲルを浸し蛋白實葡染色し、芒らにバックグラ
ンドの脱染色をし、残存している染色蛋白質スポットの
吸光度を測定する。A two-dimensional electrophoretic analysis method using polyacrylamide gel as a support, which has been conventionally used as a method for simultaneous multi-component analysis of blood proteins (for example, [Protein/Nucleic Acid/Enzyme])
, Kyoritsu Shuppan, September 1978 issue, p. 211), 1. A long slender cylindrical gel containing an ampholytic electrolyte was created in a glass tube, a voltage was applied to both ends of the Kel, and it was added to one end of the Kel. Component proteins in a blood sample are separated by their isoelectric point differences (first-dimensional separation). Next, the cylindrical Kel, which has completed the first-dimensional separation, is extruded from the glass tube onto the low concentration end of a flat Kel, which has an S degree gradient of acrylamide, which was created between two glass plates. put it on,
Electrification is again applied in the direction of the concentration gradient of the flat plate Kel, and the difference 111t due to the difference in the molecule I of the secondary round is performed. Next, in order to quantify each separated protein, the gel is placed on two glass plates that are sized I"i. For example, the gel is immersed in a dye solution such as Komatsui Blue to stain the protein. Destain the background from the awns and measure the absorbance of the remaining stained protein spots.

しかしながら、一次元目に用いる細長いケルは柔らかく
、カラス管から押し出し、平板ケルの一端にす′＠１な
く密着させる操作を機械的方法によ多行なうことは困難
である。又、二次元泳動後の柔かい平板ケルをカラス板
の間から取り出し、染色、脱染色、吸光度測定など一連
の操作中取扱うのも、ゲルの破損なとの問題があり困難
である。However, the elongated shell used in the first dimension is soft, and it is difficult to extrude it from the glass tube and bring it into close contact with one end of the flat shell using a mechanical method. Furthermore, it is difficult to remove the soft gel gel from between the glass plates after two-dimensional electrophoresis and handle it during a series of operations such as staining, destaining, and absorbance measurement because of the problem of damaging the gel.

本発明の目的は、上記欠点を改良した操作性の良い簡易
な二次元′電気泳動法を提供することにある。An object of the present invention is to provide a simple two-dimensional electrophoresis method with good operability that overcomes the above-mentioned drawbacks.

本発明Ｃ１一次元目と二次丸目の泳動に用いるケルを区
別せす、同一の平板状ケルを用いて二次元′電気泳動を
行なうものである。Invention C1 Two-dimensional 'electrophoresis is carried out using the same plate-shaped gel, which differentiates the cells used for first-dimensional and second-order round electrophoresis.

すなわち、本発明に、ガラス、又に、ポリエステルのよ
うな機械的強度の筒い基板をシランカップリング剤（Ｒ
８ｉＸｓ、Ｒは例えはヒニル基、Ｘは例えはエトキシ基
で代表される有機残基よりなる化合物）で処理し、この
基板上に、一方向にアクリルアミドの濃度勾配ｋｍする
ケル會結合、固定式ぜて作成し、ケルの低旋夏部分に被
分析試料と両性電解質の混合物を付加し、ケルの濃度一
定方向に通電し、一次元目の等電点分離を行ない、次に
、この平板ケルのケル１＃展勾配方向に通電し、分子知
゛分離を行なうものである。尚、泳動分離に用いる平板
ゲルには、あらかじめ、硫酸ナトリウム、塩化カリウム
、などの強電解ＩＸ（ｆ−含ませてお゛き、一次元目の
泳動開始時のケルの導電性を確保□　する。That is, in the present invention, a mechanically strong cylindrical substrate such as glass or polyester is coated with a silane coupling agent (R
8iXs, R is a compound consisting of an organic residue such as a hinyl group and A mixture of the sample to be analyzed and an ampholyte is added to the low-temperature part of the Kel, and electricity is applied in a direction with a constant concentration of Kel to perform first-dimensional isoelectric point separation. Electricity is applied in the direction of the Kel 1# expansion gradient to perform molecular separation. In addition, the flat gel used for electrophoretic separation is pre-impregnated with a strong electrolyte IX (f-) such as sodium sulfate or potassium chloride to ensure the conductivity of the gel at the start of the first-dimensional electrophoresis. .

以下、本発明の一実施例を図面により説明する。An embodiment of the present invention will be described below with reference to the drawings.

第１図に示すように一辺が１１ｃｍ四方のカラス板１の
片面をメタアクリルオキシプロピルトリメトキシシラン
で処理し、ガラス板上１ｏｃｒｎ四方にアクリルアミド
の濃度勾配４〜２０％を有し、かツ、塩化カリウムを含
む厚み２ｍｍの平板ケル２を作成する。矢印はポリアク
リルアミドケルの低濃度側から高濃度側への濃度勾配を
示す。このケルの低ｖ！に置部で、かつ、ケルの中央部
に６閣四方の溝３をあけ、この溝の中に血清１０μ沼と
両性電解質であるアンフオライン（ＬＫＨ社製、スウェ
ーテン、電解質濃度４０％、９８幅３．５〜１０）６０
μ石の混合物合計７０μｍ（３に加ゎえる。次に、０．
０４ＭのＮａＯＨ水溶液を含浸させだ液絡／＋３Ｐ紙４
を第１図に示したように置き、０．０４　ＭのＮａＯＨ
を含む負電極槽９に接続する。同様に、０．０１Ｍのリ
ン酸を含浸させた液絡用Ｐ紙５を０、ＯＩＭのリン酸を
含む正電極槽１０に接続し、両電極間に通寛し、血清蛋
白質を等電点差により分離する。次に、この液絡をとシ
去り、＃を除く平板ゲル全面に０．５Ｍのトリスヒドロ
キシメチルアミノメタンおよび０．３８Ｍのグリシンを
含むバッファ（以下Ａ液という）を含浸嘔せたｒ紙６を
乗せ、Ａ液を平板ゲルに浸透させる（第２図）。As shown in FIG. 1, one side of a glass plate 1 with a side of 11 cm is treated with methacryloxypropyltrimethoxysilane, and a concentration gradient of acrylamide of 4 to 20% is formed on each side of the glass plate by 1 ocrn. A flat plate 2 containing potassium chloride and having a thickness of 2 mm is prepared. The arrow indicates the concentration gradient of polyacrylamide Kel from the low concentration side to the high concentration side. This Kel's low v! At the same time, in the central part of the cell, a groove 3 of 6 squares was made, and in this groove, 10μ of serum and the ampholyte Ampholine (manufactured by LKH, Sweten, electrolyte concentration 40%, 98 width 3) were added. .5~10)60
A total of 70 μm of μ stone mixture (add to 3. Then 0.
04M NaOH aqueous solution impregnated liquid junction/+3P paper 4
was placed as shown in Figure 1, and 0.04 M NaOH
It is connected to the negative electrode tank 9 containing the negative electrode tank 9. Similarly, the liquid junction P paper 5 impregnated with 0.01M phosphoric acid is connected to the positive electrode tank 10 containing 0.01M phosphoric acid, and the fluid is passed between both electrodes, and serum proteins are Separate by Next, this liquid junction was removed, and the entire surface of the flat gel except # was impregnated with a buffer containing 0.5M trishydroxymethylaminomethane and 0.38M glycine (hereinafter referred to as solution A). onto the plate and allow Solution A to penetrate into the flat gel (Figure 2).

次に、このＰ紙を除き、ケルの高濃度部、および、極槽
１１および負電極槽１２に接続し、両電極間に通電し、
血清蛋白質を分子量差により分離する（第３図）。Next, remove this P paper, connect it to the high concentration part of Kel, the electrode tank 11 and the negative electrode tank 12, and apply electricity between both electrodes.
Serum proteins are separated based on their molecular weight differences (Figure 3).

以上、同一の平板状ゲル板を用い、等電点分離および、
分子量分離からなる二次元泳動分離を行なった。分離蛋
白Ｊを含む平板ゲルヶ、ケルを直接取扱うことなく、カ
ラス基板全取扱い、コーマソシープル−１（、−２５０
の酢酸−メタノール混液に浸し、次に、このゲルを酢酸
水浴液に浸し、蛋白質を含まないケルの部分に在存する
上記染料を除去したところ、約１００種の蛋白質の分離
スボつの機械的強度の低いゲル状支持体を直接取り扱う
必要がなくなり、固い基板ごとケル會取り扱えは良いの
で泳動分離操作は大巾に簡略化さ口た。As described above, using the same flat gel plate, isoelectric focusing and
Two-dimensional electrophoretic separation consisting of molecular weight separation was performed. Flat gel plates containing separated protein J, all glass substrates can be handled without directly handling gels, combaso sheep-1 (,-250
The gel was immersed in an acetic acid-methanol mixture, and then immersed in an acetic acid water bath to remove the dye present in the gel that does not contain proteins. Since it is no longer necessary to directly handle the low-quality gel-like support, and the gel can be handled along with the solid substrate, the electrophoretic separation operation has been greatly simplified.

また、一次元目と二次丸目のケルの接合部分が本発明に
おいてに存在しないので、蛋白質の一次元目のゲルから
二次丸目のゲルへの移動時に生じやすい分離の乱れも消
失した。Furthermore, since there is no joint between the first-dimensional and second-order gels in the present invention, disturbances in separation that tend to occur when proteins move from the first-dimensional gel to the second-order gel are eliminated.

等電点差による分離と分子量差による分離を同一ゲルで
行なう場合、等電点分離用の両性ｍ解質を予めゲルに含
ませて作成しておく方法も考えらｒしる。しかしながら
、この場合、両性電解質が保存により劣化し、分離パタ
ーンが変動し易い。病院の臨床検査室でに同一分析に袈
する仕事量は少ない方が良いので、本発明におけるよう
な強電質のみを含んた経時的に安定なゲルを別途作成し
て保存しておき随時用いる方法にメリットが大きい。If separation based on isoelectric point difference and separation based on molecular weight difference are to be performed in the same gel, it may be possible to prepare a gel by pre-containing an amphoteric electrolyte for isoelectric point separation. However, in this case, the ampholyte deteriorates upon storage, and the separation pattern tends to fluctuate. Since it is better to reduce the amount of work required for the same analysis in the clinical laboratory of a hospital, it is preferable to separately prepare a gel that is stable over time and contain only a strong electrolyte as in the present invention, and store it and use it as needed. There are great benefits to

本発明では、等電点分離のために臨床検査室が特別に行
なう仕事は血液と両性電解質を混合するという簡単にし
て再現性の良い操作のみでおる。In the present invention, the only special work required by the clinical laboratory for isoelectric point separation is the simple and reproducible operation of mixing blood and ampholytes.

両性電解質をケルに含ませる通常の方法としては、ゲル
の濃度勾配の低い部分のみに局所的に含ませる方法とケ
ル全体に含ませる方法とがあるが、前者でにゲル中での
両性！解餉の拡散が起り等電点分離パターンに乱ｊが生
ずる。後者の場会、二次元泳動後もゲル中に比較的多量
に残存する両性１解賀が蛋白鵞の検出を防害する（一般
に、蛋白個検出用試楽は婢電点分離用向性亀解質とも反
応する）。The usual methods for including ampholytes in gel are to include them locally only in areas where the concentration gradient is low, and to include them throughout the gel, but in the former case, the amphoteric electrolyte in the gel is completely absorbed! Diffusion of decoupling occurs and disturbances occur in the isoelectric point separation pattern. In the latter case, a relatively large amount of amphoteric electrolyte remaining in the gel even after two-dimensional electrophoresis prevents the detection of protein molecules (generally, the sample for protein detection is a tropic electrolyte for electrophoresis). (also reacts).

以上、種々の見地から比較しても、両性電解質と血液の
混合物１ｉ１度勾配ケルに添加して用いる本発明の方法
はメリットが太きい。As described above, even when compared from various viewpoints, the method of the present invention, which uses a mixture of ampholyte and blood by adding it to a 1/1 degree gradient cell, has significant advantages.

【図面の簡単な説明】[Brief explanation of the drawing]

第１図、第２図および第３図は本発明の方法を実施する
ための泳動装置の平面図である。 １・・・基板、２・・・泳動用支持体（ケル）、３・・
・分析試料添加用溝、４・・・液絡、５・・・液絡、６
・・・バッファ含浸ｐ紙、７・・・液絡、８・・・液絡
、９・・・負′電極槽、１０・・・正［極槽、１１・・
・正電極槽、１２・・・負電極槽。 代理人　弁理士　薄田利幸 −３０：1, 2 and 3 are plan views of an electrophoresis apparatus for carrying out the method of the present invention. 1...Substrate, 2...Migration support (kel), 3...
・Analysis sample addition groove, 4...liquid junction, 5...liquid junction, 6
...Buffer-impregnated p paper, 7...Liquid junction, 8...Liquid junction, 9...Negative electrode tank, 10...Positive [electrode tank, 11...
- Positive electrode tank, 12... negative electrode tank. Agent Patent Attorney Toshiyuki Usuda-30:

Claims (1)

【特許請求の範囲】[Claims] １、二次元電気泳動分離法において、泳動用支持体とし
て一面を基板に固定した平板状のポリアクリルアミド濃
度勾配ケルを用い、このゲルの非固定面の低濃度部分に
被分析試料と両性電解質の混合液を付加し、ケルｍ度一
定方向に通電し、試料生成分をその等電点差によ部分離
し、次に、同一ケルの濃度勾配方向に通電し試料成分を
その分子量差にもとつき分離することを特徴とする簡易
二次元電気泳動法。1. In the two-dimensional electrophoretic separation method, a flat polyacrylamide concentration gradient gel with one side fixed to the substrate is used as a support for electrophoresis, and the analyte and ampholyte are added to the low concentration part of the non-fixed side of the gel. Add the mixed solution, apply electricity in a certain direction at Kelm degrees, and separate the sample components by the isoelectric point difference, then apply electricity in the direction of the concentration gradient of the same Kel to separate the sample components based on the molecular weight difference. A simple two-dimensional electrophoresis method characterized by separation.