CN1191310A - Asymmetrical gradient electric field electrophoresis tank and electrophoresis method - Google Patents
Asymmetrical gradient electric field electrophoresis tank and electrophoresis method Download PDFInfo
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- CN1191310A CN1191310A CN96117052A CN96117052A CN1191310A CN 1191310 A CN1191310 A CN 1191310A CN 96117052 A CN96117052 A CN 96117052A CN 96117052 A CN96117052 A CN 96117052A CN 1191310 A CN1191310 A CN 1191310A
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Abstract
An electrophoresis method using an electrophoretic tank with asymmetric gradient electric field for macro-molecular separation and analysis features that in its electrophoretic tank, the gel with single-row sample slot and its supporter are arranged asymmetrically to make the one end of sample slot near the central line and another end near electrode in order to obviously increase separation effect and the resolution of biological macro-molecular band. For the double-row sample slot, the gel and its supporter are arranged in the center for reference electrophoresis of two sample groups in the gel where charge effect gradient is increased and decreased.
Description
The present invention is a kind of gel electrophoresis groove and electrophoresis method of separating bio macromolecular substances, belongs to the big molecular separation and the analysis technical field of biotechnology.
Gel electrophoresis is the common technology of biomacromolecule materials such as separation and analysing protein, nucleic acid.Gel electrophoresis apparatus is by electrophoresis tank and provide electrophoresis apparatus two parts (working simultaneously) of power supply to form for it.Existing steady direct current electrophoresis tank (horizontal strip electrophoresis groove, vertical electrophoresis groove etc.) all is that the center position between a pair of positive and negative electrode (being generally the platinum wire electrode of pair of parallel) is set gel bearing support (tabular or tubulose), during electrophoresis, separate in the gel of sample nearly all length of placing placed in the middle between positive and negative electrode.Electric Field Distribution and superposition principle according to even long straight conductor, at this gel that is arranged in the electrode pair middle position, electric field intensity is the most weak with electrophoresis midline between two electrodes (equating with effective electrophoresis distance of anodal and negative pole), strengthen gradually by two electrode direction of alignment in this, be the symmetry Gradient distribution.Therefore, in existing symmetry gradient electric field electrophoresis tank, when sample is added to gel near electrode (being generally a negative pole) end, when the gel other end carries out current stabilization or voltage stabilizing electrophoresis, electrophoresis pattern vertically shows as three sections property: at first in application of sample one side of gel, electrophoretic band is dense; And at the zone line of gel, the intensive and most adhesions of electrophoretic band are the disperse shape; At the opposite side of gel, electrophoretic band disperses, but wall scroll bands of a spectrum broad.Along with the increase of gel length and the corresponding prolongation of electrophoresis time, the problems referred to above are more obvious.Thereby the separating effect and the resolving power of gel electrophoresis have been limited.The crux that produces this problem is: in the electrophoresis process, at first since the charge effect of sample biomacromolecule material and gel to the molecular sieving effect of sample, molecule swimming less, that the quantity of electric charge is big is fast; Meanwhile, from this half section of gelling of sample cell to two interpolar electrophoresis center line, the gradient deceleration swimming that whole sample molecules weaken gradually along electric field intensity because less molecule is introduced into the gel area of low electric field intensity, thereby is at first slowed down; The quantity of electric charge is big more, and the amplitude of deceleration is also big more; Thereafter bigger molecule is then still with the fast speed swimming.As a result, have different molecular characteristic (molecular weight, molecular shape, electric charge etc.), trend various contiguous biomacromolecule bands of a spectrum separately are close mutually again.Typical situation is that two or more big molecules that molecular characterization is close are wall scroll bands of a spectrum or dispersion plating in adhesion in the process of electrophoresis center line swimming.Therefore, in preceding half section of gelling, the electric field intensity that gradient weakens has weakened the separating effect to sample biomacromolecule material such as gel molecular sieve effect greatly.On the other hand, in this half section of gelling, the gradient that the wall scroll bands of a spectrum weaken gradually along electric field intensity also obtains concentrating in various degree, thereby molecular characterization differs greatly, the bands of a spectrum of adhesion not, then presents arrowband more clearly.Cross after the electrophoresis center line, the gradient that biomacromolecule strengthens gradually along electric field intensity is again quickened to separate, make adjacent macromolecular mobility speed constantly increase gap, in the second half section gel, the charge effect that gradient strengthens significantly improves the separating effect of gel molecular sieve effect on sample.But because the curtailment of this half section of gelling is so that have the big molecule of fine difference and separate fully, particularly cross the polymolecular band of electrophoresis center line distance, still form the partly accepting and partly rejecting dispersion plating shape of multiple molecule from short (promptly be positioned at gel zone line electrophoresis center line near).Increase gel length and prolong electrophoresis time, can increase the electrophoresis distance between bands of a spectrum on the one hand, but then, because the also corresponding increase with quantity of heat production of resistance in the electrophoresis process, thereby the longitudinal diffusion of wall scroll bands of a spectrum is with shape disperse more apart from increase; And, increasing gel length and also cause electric-force gradient to strengthen, the separating effect of half section of gelling further weakens before making.Bigger is problematic in that, molecular characterization is close do not cross again the electrophoresis center line, finally be arranged in before the biomacromolecule of half section of gelling, stick in all the time and can not get in wall scroll bands of a spectrum or the dispersion plating separating.
Purpose of the present invention, be exactly at existing electrophoresis tank and the intrinsic theoretical defects of electrophoresis method, create a class and utilize the asymmetry electric field intensity gradient to separate and analyze the gel electrophoresis groove and the electrophoresis method of biomacromolecule material, with separating effect and the resolving power that significantly improves gel electrophoresis.
Asymmetry gradient electric field electrophoresis tank of the present invention and electrophoresis method comprise horizontal strip electrophoresis groove and vertical electrophoresis groove and corresponding electrophoresis method.Its technical characterictic is: 1. each organizes the separating gel of sample and the bearing support (2) of this gel, is arranged in half electric field (being the asymmetry gradient electric field) of electrophoresis center line (3) one sides between the positive and negative electrode (1).2. meet the 1. described electrophoresis tank of above-mentioned technical characterictic, the effective electrophoresis distance between its positive and negative electrode doubles in the length (10～20cm) of the gel bearing support of existing corresponding electrophoresis tank.1. and 2. described single asymmetry gradient electric field electrophoresis tank and electrophoresis method 3. meet above-mentioned technical characterictic, gel one end with single sample cell is positioned near the electrophoresis center line, the gel other end is then close with electrode (being generally anodal), one group of sample electrophoresis in the gel that electric field intensity gradient strengthens.1. and the 2. described pair of asymmetry gradient electric field leveling board electrophoresis tank and electrophoresis method 4. meet above-mentioned technical characterictic, the horizontal gel and the bearing support (2) (tabular) thereof that will have double sample cell are arranged at middle position between the positive and negative electrode, and correspondingly double in the length (10～20cm) of single sample cell gel bearing support; Two groups of sample comb slots are positioned at respectively near the end and medium line of horizontal gum-making rack (being complementary with the gel bearing support); During electrophoresis, one end of gel tool sample cell (4) and an electrode (1) (being generally negative pole) are close, another sample cell (4) correspondingly is positioned near the electrophoresis center line (3) on the gel, two groups of samples strengthen and gradient weakens half grows in the horizontal gel in electric field intensity gradient respectively, carry out the electrophoresis of equidirectional (5) simultaneously.1. and 2. described pair of asymmetry gradient electric field vertical slab electrophoresis groove 5. meet above-mentioned technical characterictic, the direct-view planimetric map of its upper and lower buffer electrode liquid bath is the nearly rectangle that waits size, last groove (6) is slightly less than down groove (7), two platinum wire linear electrodes (1) lay respectively at respectively bottom side parallel with well width in the upper and lower groove, and the height of two vertical gel bearing supports (2) and the length of last groove closely wait (about 20cm).During electrophoresis, be added in two groups of samples of two vertical panel gel upper ends, the while from top to down carries out abnormal shape according to electrophoresis in two vertical gels that electric field intensity gradient strengthens and gradient weakens respectively.
Use single asymmetry gradient electric field electrophoresis tank of the present invention and electrophoresis method, negative ion (or kation) sample separates to the nearly total length gel of anodal (or negative pole) end near the sample cell the electrophoresis center line, the beneficial effect that produces shows as: (1) is because this electrophoresis tank and electrophoresis method produce gradient enhanced charge effect, strengthen the separating effect of gel molecular sieve effect significantly, thereby close for molecular characterization in the sample, existing electrophoretic techniques is not easy to or indissociable biomacromolecule, can separate effectively; (2) various biomacromolecules even acceleration in gel separated in the sample, thereby bands of a spectrum are scattered in the total length gel equably, and resolving power significantly improves; (3) molecular weight of the sample biomacromolecule of being measured in conjunction with the SDS denaturing gel electrophoresis, linear with the migration distance of this molecule, thereby avoided existing electrophoresis method must carry out complicated process to number conversion and drawing standard molecular weight curve, significantly improve the accuracy of mensuration, and reduce requirement the reference material complicacy; (4) along with the suitable increase of gel length, the corresponding enhancing of electric-force gradient makes the dispersion degree increase of electrophoretic band, the molecular range of separating enlarge, thereby further improve separating effect; (5), be effective required gel length of separation and electrophoresis time and all shorten, thereby reduce cost and increase work efficiency than prior art for certain sample biomacromolecule potpourri.
Use of the present invention pair of asymmetry gradient electric field electrophoresis tank and electrophoresis method, sample is added in gel respectively by in the sample cell and near the sample cell the electrophoresis center line of electrode tip, can in the gel that electric field intensity gradient increases and gradient weakens, carries out special-shaped electrophoresis respectively simultaneously.Except that producing above-mentioned beneficial effect, in the gel that electric field intensity gradient weakens, molecular characterization differs greatly in the sample, NA big molecule band obtains concentrating in various degree, and the sharpness of this class band and detection sensitivity are increased.Thereby, can increase quantity of information by same sample contrasting of electrophoresis pattern in special-shaped electric field analyzed.
Fig. 1 is of the present invention pair of asymmetry gradient electric field leveling board electrophoresis tank primary structure position and electrophoresis method synoptic diagram, and the relative position and the electrophoresis direction (5) of a pair of platinum wire electrode (1), gel bearing support (2), electrophoresis center line (3) and sample cell (4) wherein arranged.Fig. 2 is the primary structure synoptic diagram of of the present invention pair of asymmetry gradient electric field vertical slab electrophoresis groove, wherein also has groove (6) and following groove (7) on the electrode buffer.
Specific embodiments of the present invention is as follows:
At the rectangle organic glass groove inner bottom part two ends of the long 44cm of an inside casing, wide 14cm, high 8cm, fixing a length respectively is the platinum wire linear electrode of 0.2mm greater than 14cm, diameter, and is connected with electrode jack outside the lead-ingroove; Apart between the end 2cm to 22cm, fix the gel bearing support of the hollow (being convenient to the water cycle cooling) of a long 20cm, wide 14cm, high 4cm, and (floorage is near 20 * 14cm to make the mobilizable horizontal gum-making rack that is complementary with it at the groove inner bottom part
2) and multiple tooth sample comb; On gum-making rack,, set the sample comb slot apart from an end 1cm place.Promptly make single asymmetry gradient electric field leveling board electrophoresis tank.Before the electrophoresis, earlier recording length in preseting the horizontal gum-making rack of sample comb is that 20cm, wide nearly 14cm, thickness are the gel slab of 3mm, after treating that gel fully solidifies, go sample comb, the gel location that makes the sample cell place is near electrophoresis center line, the close positive electrode of the gel other end between electrode; In electrophoresis tank, add suitable alkaline electrode buffer to just submergence gel level face; The connection electrode line; Energized behind the application of sample is carried out current stabilization or voltage stabilizing electrophoresis.Promptly obtain the beneficial effect of single asymmetry gradient electric field electrophoresis tank of the present invention and electrophoresis method.
Above-mentioned electrophoresis tank lengthening is 46cm, and gel bearing support (2) transform long 40cm, high 5cm as, is 3cm (promptly be positioned at groove in the middle of) apart from the groove two ends, and make match, (floorage is near 40 * 14cm for mobilizable horizontal gum-making rack
2) and two groups of multiple tooth sample comb of homotype; On gum-making rack,, set two groups of sample comb slots respectively apart from an end 1cm and 21cm place.Promptly make two asymmetry gradient electric field horizontal strip electrophoresis grooves.During glue, in preseting the horizontal gum-making rack of two groups of sample comb, record earlier the tabular gel that length is 40cm; During electrophoresis, make the sample cell (4) of distance one end 1cm on the gel be positioned at negative electrode one side, then another sample cell (4) is positioned at apart from electrophoresis center line (3) 1cm place.Two groups of identical samples carry out the horizontal strip electrophoresis of same direction (5) respectively in half long gel that electric field intensity gradient strengthens and gradient weakens, can obtain whole beneficial effect of the present invention.
Two asymmetry gradient electric field vertical slab electrophoresis grooves, the direct-view planimetric map of its upper and lower buffer electrode liquid bath is the nearly rectangle that waits size, last groove (6) is slightly less than down groove (7), last groove housing is 20cm * 18cm * 3cm, wherein, the notch of 16cm * 2cm is set in the 18cm * 3cm end frame upper limb central authorities at last groove two ends; Following groove inside casing is 22cm * 20cm * 4cm.Two platinum wire electrodes (1) lay respectively at each parallel with well width in upper and lower groove side; The height (the 2cm notch degree of depth that contains groove end frame) on two vertical gel bearing support (2) (tabular) planes is 21cm.Its gum-making rack and sample comb structure, glue and add all similar prior art of quadrat method adopt discontinuous gel electrophoresis system (contain and concentrate glue 3cm and separation gel).During electrophoresis, be arranged in two groups of negative ion (or kation) sample of two vertical panel gel upper end sample cells, to anodal (or negative pole) direction, the while from top to down carries out special-shaped electrophoresis in two vertical gels that electric field intensity gradient strengthens and gradient weakens respectively by negative pole (or anodal).Can obtain whole beneficial effect of the present invention.
Claims (5)
1. a separation and steady direct current electrophoresis tank and the electrophoresis method of analyzing the biomacromolecule material, it is characterized in that: the separation gel of each group sample and the bearing support (2) of this gel, half electric field that is arranged in electrophoresis center line (3) one sides between the positive and negative electrode (1) is the asymmetry gradient electric field.
2. electrophoresis tank according to claim 1 and electrophoresis method is characterized in that: the electrophoresis distance between positive and negative electrode doubles in the length (10～20cm) of existing electrophoresis tank gel bearing support.
3. according to claim 1 and 2 described single asymmetry gradient electric field electrophoresis tank and electrophoresis methods, comprise horizontal strip electrophoresis groove and vertical electrophoresis groove and corresponding electrophoresis method, it is characterized in that: gel one end with single sample cell (4) is positioned near the electrophoresis center line, and the gel other end and electrode (being generally anodal) are close; One group of sample electrophoretic separation in the gel that electric field intensity gradient strengthens.
4. according to claim 1 and the 2 described pairs of asymmetry gradient electric field horizontal slab gel electro-phoresis grooves and electrophoresis method, it is characterized in that: leveling board gel and bearing support (2) thereof with double sample cell, be arranged at the middle position between the positive and negative electrode (1), and double in the length (10-20cm) of single sample cell gel bearing support, the slot of two groups of sample comb is positioned at respectively near the end and medium line of horizontal gum-making rack (being complementary with the gel bearing support); During electrophoresis, a sample cell of gel end (4) and an electrode are close, another sample cell (4) then is positioned between electrode near the electrophoresis center line (3) on the gel, and two groups of samples are electrophoresis in half long gel that electric field intensity gradient strengthens and gradient weakens respectively with carrying out horizontal direction simultaneously.
5. according to claim 1 and the 2 described pairs of asymmetry gradient electric field vertical slab gel electrophoresis grooves and electrophoresis method, it is characterized in that: the direct-view planimetric map of upper and lower buffer electrode liquid bath is the nearly rectangle that waits size, last groove (6) is slightly less than down groove (7), two platinum wire electrodes (1) lay respectively at respectively bottom side parallel with well width in the upper and lower groove, and the height of two vertical gel bearing supports (2) and the length of last groove are near etc.; During electrophoresis, be positioned at two groups of samples of two vertical panel gel upper ends, respectively while from top to down electrophoresis in two vertical gels that electric field intensity gradient strengthens and gradient weakens.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96117052A CN1191310A (en) | 1996-08-08 | 1996-08-08 | Asymmetrical gradient electric field electrophoresis tank and electrophoresis method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN96117052A CN1191310A (en) | 1996-08-08 | 1996-08-08 | Asymmetrical gradient electric field electrophoresis tank and electrophoresis method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100342103C (en) * | 2002-03-28 | 2007-10-10 | 王杏林 | Architecture veneer and assembling process thereof |
| CN101124477B (en) * | 2004-12-31 | 2011-11-09 | 迪米特里奥斯·赛德里斯 | Electrophoresis method and device for separating objects |
| CN102288668A (en) * | 2011-07-26 | 2011-12-21 | 吉林大学 | Electrophoresis tank using electroconductive diamond film as electrode |
| CN102325786A (en) * | 2009-02-23 | 2012-01-18 | 生物辐射实验室股份有限公司 | Electroblotting cassette with manually releasable electrodes of adjustable spacing |
-
1996
- 1996-08-08 CN CN96117052A patent/CN1191310A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100342103C (en) * | 2002-03-28 | 2007-10-10 | 王杏林 | Architecture veneer and assembling process thereof |
| CN101124477B (en) * | 2004-12-31 | 2011-11-09 | 迪米特里奥斯·赛德里斯 | Electrophoresis method and device for separating objects |
| CN102325786A (en) * | 2009-02-23 | 2012-01-18 | 生物辐射实验室股份有限公司 | Electroblotting cassette with manually releasable electrodes of adjustable spacing |
| CN102325786B (en) * | 2009-02-23 | 2014-06-18 | 生物辐射实验室股份有限公司 | Electroblotting cassette with manually releasable electrodes of adjustable spacing |
| CN102288668A (en) * | 2011-07-26 | 2011-12-21 | 吉林大学 | Electrophoresis tank using electroconductive diamond film as electrode |
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