DD152423A1 - ELECTROTRANSFER OF NUCLEIC ACIDS OR PROTEINS FROM DISCONNECTED TO ACTIVATED PAPERS - Google Patents

Abstract

The invention relates to a method for the rapid transfer of nucleic acids or proteins from separating gels to paper activated with cyanuric acid chloride by means of electrophoresis. The method is suitable for affinity chromatographic investigations in biochemical and medical research. It is a surface electrode used, which consists of a plastic box with finely perforated bottom, the bottom of which is poured with gel (agarose, polyacrylamide).

Description

Dr. H.-D. Hunger H * Grützmann

"Electro-transfer of nucleic acids or proteins from separating fell on activated papers"

t the invention

The invention relates to a method for electrotransfer of charged compounds, in particular of nucleic acids, proteins to activated papers, which finds application in biochemistry and medicine for affinity chromatographic investigations.

Characteristic erist ik of the known technical solutions The transfer of nucleic acids on with diazo gr £ ppen activated paper is known. In this case, the elution of the nucleic acids from the gel onto the filter paper takes place through the buffer flow. (Southern, Jo Mol. Biol. 98 (1975) 503-517). But very long transfer times are required, and there is an inactivation of the paper "^:;

Another method for rapid transfer of nucleic acids to diaso-group activated paper (DBM) uses concentrated saline solutions and requires depurination of the nucleic acids. (Wahl et al PNAS £ 6 (1979) 3683-3687). The transmission takes 2-4 hours. In addition, the required weight loading of the gel causes distortion of the bands,

Electro-radical apparatuses with vertical suspension of the gel in buffer chambers US Pat. 3,989,612 have much more elaborate constructions (Bittner, Anal, Biochem 102 (1980) 459,471). There is a "risk of band distortion during transfer." In addition, a very high current (2 A) must be created *

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• Object of the invention

The aim of the invention is to simplify the transfer of charged compounds, such as nucleic acids, proteins on activated paper and perform in less time.

Explanation of the essence of the invention

The object of the invention is to develop a method for achieving a rapid transfer of gels and nucleic acids separated on gels to activated papers without band distortion.

The object is achieved by a method for the transfer of nucleic acids or proteins to activated paper according to the invention by placing the separating gel on layers of thick filter paper (PH 18) moistened with electrode buffer of a stand provided with a nylon net and in the buffer-filled electrophoresis vessel. whereby the moistened layers are in direct contact with the electrode buffer

to have. The nucleic acids or proteins are by applying

s

a DC voltage by means of a surface electrode on the directly on the separating gel, activated paper transferred. As activated paper, a paper treated with cyanine chloride is advantageously used

 The surface electrode used is expediently designed as .Plastebehälter with finely perforated bottom. The bottom of the plastic container is filled with 1% agarose about 2 cm thick, while the overlying space is filled with Slektrophoresepuffer. In this room, a platinum electrode is hung.

The inventive method requires less equipment and allows a faster transfer of the bands of ITukleinsäuren (15 min) · There is no band distortion

 The process requires less technical effort *

Auaführunffsbeisp i el

The Errungungögemäße method is explained in more detail below with reference to the apparatus shown »Ea Pig ,: 1 the total apparatus for transfer Fig.i 2 the surface electrode.

An electrophoresis vessel 1 is filled with electrophoresis buffer (0.15 M phosphate buffer) to the level of an ITyl network secured as a top to a pedestal 4 located in the electrophoresis vessel 1. Three layers of moistened filter paper 5 (FN 18) are placed on the IJylonnetz and then the separating gel б is placed. On the prepared separating gel б (1.5 % agarose for KNS separations) 'directly activated the transfer paper 7 (cyanuric chloride paper) and placed on this one layer of filter paper 5 (FlI 18), RlIS-GeIe be 2 χ .15 Washed at room temperature in 0.05 K HaOH and then incubated with 0.15 M phosphate buffer pH 6.

DNA gels are washed for 30 minutes in 0.5 M HaOH, 1.5 M HaCl and then incubated with 0.15 M phosphate buffer pH b. On the filter paper 5 (Ш 18) to set a surface electrode, which is designed as a plastic container 8 with perforated bottom. The bottom of the plastic container 8 is about 2 cm high with 1 folgev agarose 9 poured »The overlying electrophoresis buffer space 10 v / ird filled with electrophoresis buffer 2 and suspended in this an electrode 3. The anode used is a platinum wire loop (4 cm). Then you apply the DC voltage to the electrodes 3 »The size of the surface electrode depends on the size of the separating gel. By applying the DC voltage, the compounds separated in the separating gel are moved out of the gel into the activated paper, where they are covalently bonded to the paper. The 'transfer of z' B. rHNS, tRITS, 9 S globin mMS is complete at -60 V, 200 mA in 15 minutes at 4 ° G.

Claims (3)

Erfindungsansprach 1 "method for the transfer of nucleic acids and proteins from separating gels on activated paper, characterized in that the transfer by electrophoresis by the separating gel on moistened filter paper layers einos provided with Uylonnetz, located in the buffer-filled Slektrophoresegefäß stator is placed and the Hukleinsäuren or proteins by a flat electrode on the activated paper located directly on the separation gel v / earth. 2, method according to item 1, characterized in that a paper treated with cyanuric acid chloride is used as the activated paper » 3 Method according to item 1, characterized in that a surface electrode is used, which consists of a two-to-three-centimeter plastic gel (agarose, polyacrylamide) with finely perforated bottom "» / j. String drawing