CN1051245C - Preparation type isoelectric point electrophoresis separating method and equipment - Google Patents
Preparation type isoelectric point electrophoresis separating method and equipment Download PDFInfo
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- CN1051245C CN1051245C CN95101184A CN95101184A CN1051245C CN 1051245 C CN1051245 C CN 1051245C CN 95101184 A CN95101184 A CN 95101184A CN 95101184 A CN95101184 A CN 95101184A CN 1051245 C CN1051245 C CN 1051245C
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Abstract
The present invention relates to a preparation type isoelectric point electrophoresis separation method and separation plant for separating different components of protein, which belongs to the technical field of biological chemical engineering. In the method, a gel film is arranged between two parallel long narrow electrodes along a length direction, and the space between the electrodes is divided into a middle separation chamber and two washing chambers on both sides. A sample solution to be separated with a pH value regulated to a isoelectric point (a pI value) of a target product is led in the separation chamber, a common buffer solution with a pH value which is the isoelectric point of the target product is led in the washing chambers, and the isoelectric point and hybridprotein which is different from the target product are transferred in the washing chambers on both of the two sides under the action of an electric field to be washed out. Consequently, the separation is realized.
Description
The present invention relates to a kind of preparation type isoelectric point electrophoresis separating method that is used to separate both sexes biological substance (as protein, polypeptide), belong to technical field of biochemical industry.
In prior art, the isoelectric focusing device that a kind of multi-cavity chamber is arranged, name is called " isoelectric focusing in the electrophoretic apparatus of multi-cavity chamber ", come from one piece of paper that is called " Large-scale fracitionationof proteins by isoelectric focusing in a multi-compartment electrolysisapparatus " of being write by agate thatch Jonathan (Mats.Jonsson) and Harry Li Bo (Harry.Rilbe), be published in " electrophoresis " magazine first phase in 1980 the 3rd page to the 14th page (Electrophoresis, 1, P3-14 (1980)).The continuous pH gradient fields that this technology utilizes carrier ampholyte solution to form is background, focuses under the corresponding pH background that do not coexist of protein according to isoelectric point separately and is separated, and proposed corresponding separation equipment.Its core is an elongated cylindrical electrophoresis tank, by phase barrier film across a certain distance it is divided into the dozens of chamber, shown in accompanying drawing one: before the separation, carrier ampholyte solution is evenly mixed each chamber of back injection with treating the branch protein example; During separation, two ends at electrophoresis tank add DC voltage, electromigration takes place in carrier ampholyte under electric field action, the gradient that formation is raise to negative pole pH value gradually by positive pole, protein can not keep electroneutral in being different from the pH environment of its isoelectric point, thereby under electric field action electrophoretic migration takes place, pass barrier film, in the chamber of the pH value that is equivalent to its isoelectric point, focus on, the heat that electrophoresis produces is taken away by the complicated interior circulating cooling system of a cover, through after considerable time (2-3 days), focus on and finish like this, the albumen with different isoelectric points is separated from each other in different chambers; Open the sample export of each chamber at last, collect sample liquid respectively, in some or adjacent with it several chambers, just obtain sublimed target protein component.
The shortcoming of this technology is: (1) needs a large amount of expensive carrier ampholytes, also needs in addition to remove the carrier ampholyte that mixes in the target components sample by methods such as dialysis in subsequent treatment, makes the cost of separation very high; (2) because the migration distance of protein is very long, and electrode spacing is big, and improve the restriction that field intensity is subjected to the exchange capability of heat of auto levelizer, cause this method disengaging time long, treating capacity is lower.
In prior art, what also have a kind of multi-cavity chamber circulates the isoelectric focusing device, name is called " using the isoelectric focusing of non-carrier ampholyte buffer ", come from one piece of paper that is called " Isoelectric focusing using non-amphoteric buffers in freesolution:II.Apparatus and measures of pH stability " of being write by Pi Aier Tomi Ungerer (Pierre.Wenger) and Fei Lipu Jeff spy (Philippe.Javet), be published in 1986 the 13rd phases of " biochemistry and bio-physical method " magazine the 275th page to the 287th page (Journal of Biochemical and Biophysical Methods, 13, P275-287 (1986)).This technology adopts the form that circulates of multi-cavity chamber, utilizes pH gradient that simple buffering liquid (not containing carrier ampholyte) forms under the electric field action background as isoelectric focusing, in order to amino acid separation and protein, and has proposed corresponding separation equipment.Shown in accompanying drawing two: 1 is the electrophoresis tank of a multi-cavity chamber, and 2 is the agarose-gel film of fabric-supported, and 3 is amberplex, and 4 is heat exchanger, and 5 is the electrode solution storage tank, and 6 is the buffer solution storage tank.Gel mould and amberplex are separated into electrophoresis tank some chambers and two electrode chambers that are parallel to each other, in electrode chamber, feed electrode solution, feed acetic acid-sodium-acetate buffer in each middle chamber, electrode solution and buffer solution all circulate in separate path, both sides at electrophoresis tank load DC voltage, under electric field action, a part of ion generation electrophoretic migration in the buffer solution, enter contiguous chamber by gel mould and amberplex, after after a while, the pH value of each chamber changes, and forms the pH gradient that raises gradually from the negative electrode to the anode, and uses the background of this pH gradient as isoelectric focusing.
The shortcoming of this technology is: the pH gradient that (1) forms is stable inadequately, and the pH value of each chamber is drifted about easily, is difficult to the pH value of proofreading and correct each chamber with easy method; (2) raising of its separation accuracy is subjected to the restriction of chamber number, and too much chamber number will bring great inconvenience to operation, so can only be used for rough segmentation, is difficult to separate the approaching sample of isoelectric point.
The objective of the invention is to propose a kind of novel electrophoresis separating method, use the principle of isoelectric focusing, use simple buffering liquid to form a stable single pH value, rather than a stable p H gradient its objective is and separate a target product rather than a series of product as the background of separating.
The object of the present invention is achieved like this: between vertical two long narrow electrodes that are parallel to each other placing, along the length direction of electrode two to four gel moulds that be arranged in parallel, they are separated into three to five chambers that are parallel to each other with the space between the electrode, middle chamber is the separation chamber, the both sides of separation chamber are developing room, separate when carrying out, feed the sample liquid to be separated that the pH value transfers to the isoelectric point (pI value) of target protein in the separation chamber, feed in the developing room of both sides that pH value equals the target protein isoelectric point and electricity is led little buffer solution, load the constant DC pressure between two electrodes.
Below in conjunction with accompanying drawing, introduce content of the present invention in detail.
Accompanying drawing three is preparation type isoelectric point electrophoresis separating method schematic diagrams.
7 is electrode among the figure, 8 is gel mould, 9 sample liquid for feeding, 9a is the separation chamber, 10 buffer solutions for feeding, 10a is a developing room, 11 is target protein, 12 is the foreign protein of isoelectric point (pI value) greater than the isoelectric point of target protein, and 13 is the foreign protein of isoelectric point (pI value) less than the isoelectric point of target protein.
Its separation process is: the 9a of separation chamber feeds the sample liquid to be separated that the pH value transfers to the isoelectric point (pI value) of target protein, and developing room 10a feeds the buffer solution that the pH value equals the target protein isoelectric point, loads constant DC between two electrodes 7 and presses.Target protein molecule 11 under such pH value condition (representing with little garden circle in the accompanying drawing three) is electroneutral generally, and therefore electrophoretic migration does not take place under electric field action; The then positively charged with this understanding or negative electrical charge of the differentiated foreign protein of other isoelectric point and target protein, the protein 12 positively charged (representing with triangle in the accompanying drawing three) that isoelectric point (pI value) is big, the albumen 13 electronegative (representing with little black box in the accompanying drawing three) that isoelectric point is little, under electric field action, electrophoretic migration must take place, the gel mould that they pass the both sides, separation chamber respectively enters the both sides developing room, thereby has realized and being separated from each other of target protein.
Purpose of the present invention also can realize like this: between vertical two long narrow electrodes that are parallel to each other placing, along the length direction of electrode two gel moulds that be arranged in parallel, two amberplexes of arranged outside at gel mould, thereby the space between the electrode is separated into five chambers that are parallel to each other, intermediate cavity is the separation chamber, two chambers adjacent with the separation chamber are developing room, two chambers near electrode are electrode chamber, separate when carrying out, feed the sample liquid to be separated that the pH value transfers to the isoelectric point of target protein in the separation chamber, feeding pH value in the developing room of both sides equals the target protein isoelectric point and electricly leads little buffer solution, feed electrode solution in the electrode chamber, load constant DC between two electrodes and press.
The present invention uses ordinary buffer liquid, and as trishydroxymethylaminomethane-twenty propylhomoserin, trishydroxymethylaminomethane-acetic acid, acetic acid-sodium acetate etc., concentration is the 0.005-0.1 mol; The flow velocity of separation chamber be the 100-1000 milliliter/hour, the flow velocity of developing room be the 200-2000 milliliter/hour; The thickness of used gel mould is the 0.2-2 millimeter; Voltage between the electrode is the 20-500 volt, and current strength is not higher than 100 milliamperes, in this scope, can suitably adjust electrode voltage.
On the basis of the principle of the invention, designed the equipment of preparation type isoelectric point electrophoresis, its core all is an electrophoresis tank.
Accompanying drawing four is assembling schematic diagrames of three chamber electrophoresis tanks of preparation type isoelectric point electrophoresis.
Last figure is a plan view, figure below is a top view, 14 is the separation chamber among the figure, and 15 is developing room, and 16 is separation chamber's chamber frame, 17 is the developing room chamber body, 18 is gel mould, and 19 is conduit, and 20 is the clamping device that forms with the sticking system of lucite, 21 are supply socket, and 22 for being used for fixing the hex bolts of chamber body.
Four bolts are used for fixing chamber body 17, chamber frame 16 and gel mould 18 chamber with sealing around surrounding three, the centre is separation chamber 14, both sides are developing room 15, separation chamber and developing room all have the conduit for sample liquid and buffer solution discrepancy up and down, platinum electrode is placed in the developing room, and be connected with supply socket, electrophoresis tank is easy to dismounting and assembling, so that change gel mould and add arrangement.
Accompanying drawing five is chamber body and chamber frame schematic diagrames of electrophoresis tank.
23 is separation chamber's chamber frame, and 24,25 is both sides developing room chamber body (the two left-right symmetry), and 26 for being used for installing additional the through hole of supply socket, and 27 is platinum electrode, and 28 is the polytetrafluoroethylene conduit, and chamber body is made with insulating materials such as lucite.
Accompanying drawing six is structural representations of five chamber electrophoresis tanks of preparation type isoelectric point electrophoresis
29 is gel mould among the figure, and 30 is amberplex, and 31 is electrode, and 32 is the separation chamber, and 33 is developing room, and 34 is the anode chamber, and 35 is cathode chamber, and 36 is sample liquid, and 37 is buffer solution, and 38 is anode electrode liquid, and 39 is cathode electrode liquid.
Shown in accompanying drawing six left figure, during operation, feed the sample liquid that the pH value equals the target components isoelectric point in the separation chamber 32, feed the buffer solution that the pH value equals the target components isoelectric point in the developing room 33, feed anode electrode liquid in the anode chamber 34, feed cathode electrode liquid in the cathode chamber 35.
Owing to use amberplex and electrode solution more complicated, also can adopt the structure shown in the accompanying drawing six right figure for simplicity, four films all adopt gel mould, feed the buffer solution identical with developing room in the electrode chamber of both sides, also can play the effect of stablizing the pH value preferably.
Accompanying drawing seven is assembling schematic diagrames (plan view has only drawn) of five chamber electrophoresis tanks of preparation type isoelectric point electrophoresis
Surround five chambers of sealing all around with bolting chamber body, chamber frame, gel mould and amberplex, the centre is the separation chamber, and the both sides of separation chamber are developing room, the both sides electrode chamber of outermost (anode chamber and cathode chamber).Each chamber the conduit of coming in and going out all arranged up and down for liquid, platinum electrode is placed in the electrode chamber, and is connected with supply socket.
On the basis of the electrophoresis tank of above introduction, designed a series of preparative electrophoresis separation equipment.Make introductions all round below:
Accompanying drawing eight is schematic flow sheets of the working condition of preparation type isoelectric point electrophoresis three chambers.
40 is electrophoresis tank, and 41 is heat exchanger, and 42 is active carbon adsorption column, 43 is chuck chilled buffer storage tank, and 44 are chuck cooling sample liquid storage tank, and 45 be to organize constant flow pumps (among the figure with a signal) more, 46 is magnetic stirring apparatus, and 47 is the dc constant voltage power supply, 48 constant temperature circulators for the band refrigeration.When carrying out electrophoretic separation, the logical constant DC that goes up is pressed on the platinum electrode in two developing rooms; Sample liquid enters the 40a of separation chamber of electrophoresis tank from sample liquid storage tank 44 by the driving of constant flow pump 45, in electrophoresis tank, foreign protein is moved among the developing room 40b of both sides by gel mould under electric field action, the buffer solution that flows out from two developing rooms mixes mutually, exhaust foreign protein through entering active carbon adsorption column 42 after heat exchanger 41 heat exchange, return buffer solution storage tank 43, send into developing room by constant flow pump again and reuse; The sample liquid that process is separated flows out from the 40a of separation chamber of electrophoresis tank, enters sample liquid storage tank 44 through heat exchanger 41, enters the separation chamber of electrophoresis tank again through the driving of constant flow pump, and the straight qualified target protein product that obtains so circulates repeatedly; By the low-temperature circulating water (0-10 ℃ is adjustable) that constant temperature circulator 48 produces, in heat exchanger and each storage tank, give buffer solution and sample liquid with sufficient cooling, guaranteed the activity of biological substance; Many group constant flow pumps 45 drive buffer solution and sample liquid circulates with the flow of setting respectively; Also have the situation of change of the pH value in pH electrode (omitting among the figure) test sample liquid storage tank and the buffer solution storage tank in addition, after process was long-time, the pH value of sample liquid storage tank and buffer solution storage tank can slightly change, and can drip the adjustment of pH buffer solution this moment.
When the electrophoresis tank of three gel moulds of use and four gel moulds (constituting four chambers and five chambers respectively), the flow process of employed workflow and three chamber electrophoresis tanks recited above is basic identical.
Accompanying drawing nine is schematic flow sheets of the working condition of preparation type isoelectric point electrophoresis five chambers
49 is five chamber electrophoresis tanks, 50 is anode electrode liquid storage tank, 51 is cathode electrode liquid storage tank, and 52 is heat exchanger, and 53 is active carbon adsorption column, 54 is chuck chilled buffer storage tank, 55 are chuck cooling sample liquid storage tank, and 56 be to organize constant flow pumps (among the figure with a signal) more, and 57 is magnetic stirring apparatus, 58 is the dc constant voltage power supply, 59 constant temperature circulators for the band refrigeration.Sample liquid circulates between sample liquid storage tank 55 and the 49a of electrophoresis tank separation chamber, and buffer solution circulates between buffer solution storage tank 54 and electrophoresis tank developing room 49b, and electrode solution circulates between electrode chamber 49c and electrode solution storage tank 50,51.
Need to prove, accompanying drawing the eight, the 9th, isoelectric point electrophoretic apparatus three chambers and the typical working condition of five chambers, it is more stable to be suitable for target product, and content is not very rare situation in sample liquid.In actual applications, run into the product of very easy inactivation and a large amount of dilute concentration samples sometimes, can adopt the mode of operation of one way, shown in accompanying drawing ten, be the workflow of only drawing for simplicity and constituting by three chamber electrophoresis tanks.
Accompanying drawing ten is the isoelectric point electrophoresis workflows that are suitable for easy inactivation sample and a large amount of dilute concentration sample situations.
The 60th, electrophoresis tank (for the situation of three chambers that only drawn for simplicity), the 61st, chuck cooling sample liquid storage tank, 62 is chuck chilled buffer storage tank, the 63rd, chuck cooling sample liquid feeder, 64 is heat exchanger, 65 is active carbon adsorption column, 66 is many group constant flow pumps (among the figure with a signal), 67 is magnetic stirring apparatus, and 68 is the dc constant voltage power supply, 69 constant temperature circulators for the band refrigeration.
When electrophoresis carried out, sample liquid had not been through returning sample liquid storage tank 61 behind the electrophoresis tank 60, but entered sample liquid feeder 63, after the sample liquid of sample liquid storage tank 61 is all by electrophoresis tank 60, just finished the operation of an one way.At this moment, the sample liquid in the sample liquid feeder 63 is shifted back sample liquid storage tank 61, just can enter next one way separation process,, just can finish separation through (being generally 2-4 time) after the one way several times.The total disengaging time of this mode of operation can shorten greatly, is more conducive to guarantee the activity of biological sample, but operation is slightly more complicated than cycling.
When adopting the electrophoresis tank of three gel moulds, four gel moulds, its flow process is identical with the workflow of top two gel moulds.When adopt using the electrophoresis tank of amberplex, its flow process is basic identical, and institute's difference is to increase by two electrode solution storage tanks, and electrode solution circulates between the electrode chamber of electrophoresis tank and electrode solution storage tank.
Because the isoelectric point electrophoresis carries out in the process, still have a spot of target product to enter developing room by gel mould by effects such as diffusions, so be worth very expensive sample for separating,, designed the operating process of recyclable target product for improving yield to greatest extent.
Accompanying drawing 11 is the recyclable operational flowchart of isoelectric point electrophoresis
70 is electrophoresis tank, the 71st, and chuck chilled buffer storage tank, the 72nd, buffer solution feeder, 73 is heat exchanger, 74 are chuck cooling sample liquid storage tank, and 75 be to organize constant flow pumps (among the figure with a signal) more, and 76 is magnetic stirring apparatus, 77 is the dc constant voltage power supply, 78 constant temperature circulators for the band refrigeration, the 79th, constant flow pump, the 80th, ion exchange column, the 81st, gradient mixer, the 82nd, automatic fraction collector.
In this operating process, the buffer solution of the outlet of developing room does not return original buffer solution storage tank 71, collects with a buffer solution feeder 72 in addition.Then this part buffer solution is carried out ion-exchange chromatography, on a small quantity the target product that enters developing room by diffusion can be adsorbed by ion exchange column 80, when ion exchange column absorption when saturated, carry out gradient elution, collect the elution fraction that comprises target product in the eluent, adjust pH value and electricity and lead back (available dialysis), carry out the isoelectric point electrophoresis once more.
When adopting the electrophoresis tank of three gel moulds, four gel moulds, its flow process is identical with the workflow of top two gel moulds.When adopt using the electrophoresis tank of amberplex, its flow process is basic identical, and institute's difference is to increase by two electrode solution storage tanks, and electrode solution circulates between the electrode chamber of electrophoresis tank and electrode solution storage tank.
Effect of the present invention is to be used as the necessary pH background of isoelectric focusing owing to utilize simple buffering liquid to form a stable p H point (rather than continuous excessively district's band) in the separation process, has not only reduced cost but also avoided product is introduced new pollution.Compare with traditional multi-cavity chamber isoelectric focusing, the raising of its separation accuracy is not to realize by the number that increases chamber, but adopts several chambers (being generally three to five) of minority, just can finish the separation of degree of precision; Direction of an electric field is vertical mutually with the direction of flushing current-carrying in the separation process, can be by shortening distance between electrodes, under the low-voltage condition, produce high electric-field intensity, accelerated the component migration velocity, shortened electromigratory path, reduce the electrophoresis fuel factor, improved the efficient of separating generally; And simple in structure, the scale that is easy to carry out is amplified.
By to ox blood red-separating experiment of cow's serum pattern protein mixture and practical systems such as urokinase, interleukin-6, proved that preparation type isoelectric point electrophoresis can quick, high-precision separation purpose sample.
Introduce several embodiments of the present invention below.
Embodiment 1:
Typical process clastotype protein mixture with the isoelectric point electrophoresis
1 developing room buffer solution: acetic acid-sodium acetate (0.01 mol)
2 separation chamber's sample liquid:
Contain BHb 1 mg/ml, bovine serum albumin 1 mg/ml, use
0.01 mol acetic acid-sodium acetate preparation
3 gel mould thickness: 1 millimeter
Flow process shown in the 4 operation accompanying drawings eight
Electrode voltage: 100 volts of current strength: less than 100mA
Flow: 200 milliliters/hour of separation chambers, 200 milliliters/hour of developing rooms
5 results:
When the pH of buffer value was 4.83 (cow's serum isoelectric points), obtaining purity was 98%
Bovine serum albumin, yield 92%; The pH of buffer value is that 6.90 (ox blood is red etc.
The electricity point) time, acquisition purity is 95% BHb, and yield is greater than 90%.
100 milligrams of albumen for the treatment of capacity/hour
Embodiment 2:
Typical process clastotype protein mixture with the isoelectric point electrophoresis
1 developing room buffer solution: acetic acid-sodium acetate (0.1 mol)
2 separation chamber's sample liquid:
Contain BHb 1 mg/ml, contain bovine serum albumin 1 mg/ml, use
0.1 mol acetic acid-sodium acetate preparation
3 gel mould thickness: 0.2 millimeter
Flow process shown in the 4 operation accompanying drawings eight
Electrode voltage: 20 volts of current strength: less than 100mA
Flow: 100 milliliters/hour of separation chambers, 5000 milliliters of/hour 5 results of developing room:
When the pH of buffer value was 4.83 (cow's serum isoelectric points), obtaining purity was 88%
Bovine serum albumin, yield 71%; The pH of buffer value is that 6.90 (ox blood is red etc.
The electricity point) time, acquisition purity is 79% BHb, yield 60%.Handle
Contain BHb 0.5 mg/ml, contain 0.5 milligram of bovine serum albumin/in the least
Rise, prepare 3 gel mould thickness with 0.01 mol acetic acid-sodium acetate: 2 millimeter 4 yin, yang amberplex: flow process shown in 0.5 millimeter 5 operation accompanying drawing nine
Electrode voltage: 350 volts of current strength: less than 100mA
Flow: 500 milliliters/hour of separation chambers, 1000 milliliters/hour of developing rooms; Anode
The sulfuric acid solution of chamber 0.05 mol, 300 milliliters/hour; Cathode chamber 0.1
The sodium hydroxide solution of mol, 300 milliliters of/hour 6 results:
When the pH of buffer value was 6.90 (the red isoelectric points of ox blood), obtaining purity was 95%
BHb, yield is greater than 95%.50 milligrams of albumen for the treatment of capacity/hour embodiment 4: separate interleukin-6 1 electrode voltage with recyclable flow process: 155 volt of 2 developing room buffer solution: trishydroxymethylaminomethane-citric acid (pH=5.65,0.01 mol) 3 separation chamber's sample liquid:
Through the interleukin-6 inclusion body of rough segmentation broken liquid, wherein interleukin 8
About 0.22 mg/ml of plain-6 content is with trishydroxymethylaminomethane-lemon
Lemon acid (pH=5.65,0.01 mol) dialysis equilibrium 4 gel mould thickness: 1.5 millimeter 5 operation accompanying drawing 11 reclaimer operation flow process
Electrode voltage: 155 volts of current strength: less than 100mA
Flow: 300 milliliters/hour of separation chambers, 500 milliliters of/hour 6 recovery of developing room:
Ion exchange column: DEAE-Sephadex A-50 anionite
Recovery sample electrophoresis: 140 volts of electrode voltages, 400 milliliters of separation chamber's flows/little
The time, 2000 milliliters of/hour 7 results of developing room flow:
After primary sample and recovery sample mixing, SDS-poly-third is carried out in sampling
The electrophoretic analysis of alkene acid amides with standard control, does not have the impurity bands of a spectrum to detect.Total egg
White yield is greater than 80% embodiment 5: one-pass operation separates urokinase 1 developing room buffer solution:
Trishydroxymethylaminomethane-glycine (pH=8.60,0.02 mol) 2 separation chamber's sample liquid:
Urokinase half elaboration (is 4000 unit of activity/milligram albumen than work) is used three hydroxyls
Methylamino methane-glycine (pH=8.60,0.02 mol) dialysis is flat
3 gel mould thickness weigh: 1.5 millimeter 4 operation accompanying drawing ten one-pass operation flow process
Electrode voltage: 180 volts of current strength: less than 100mA
Flow: 200 milliliters/hour of separation chambers, 500 milliliters/hour of developing rooms
Four one-pass operation 5 results:
Obtain to live than the urokinase elaboration of living greater than 40000 unit of activity/milligram albumen
The property yield be 32% embodiment 6: one-pass operation separates urokinase 1 developing room buffer solution:
Trishydroxymethylaminomethane-glycine (pH=8.60,0.005 mol) 2 separation chamber's sample liquid:
Urokinase half elaboration (is 4000 unit of activity/milligram albumen than work) is used three hydroxyls
Methylamino methane-glycine (pH=8.60,0.005 mol) dialysis is flat
3 gel mould thickness weigh: 1.5 millimeter 4 operation accompanying drawing ten one-pass operation flow process
Electrode voltage: 500 volts of current strength: less than 100mA
Flow: 1000 milliliters/hour of separation chambers, 1000 milliliters/hour of developing rooms
Three one-pass operation 5 results:
Obtain to live than the urokinase product of living greater than 20000 unit of activity/milligram albumen
The property yield is 40%
Claims (14)
1, a kind of electrophoresis tank that is used for the isoelectric point electrophoretic separation of preparation type is characterized in that in the chamber body of electrophoresis tank, and two long narrow electrodes vertically are set; Between described two electrodes, along the electrode length direction gel mould that be arranged in parallel; Described gel mould is separated into several chambers that are parallel to each other with the space between the described electrode, and middle chamber is the separation chamber, in the separation chamber, feeds the sample liquid to be separated that the pH value transfers to the isoelectric point of target protein; Both sides in the separation chamber are developing room, and in developing room, feeding pH value equals the target protein isoelectric point and electricity is led little buffer solution; Between described two electrodes, load constant DC and press, the chamber at electrode place is cloudy (sun) utmost point chamber.
2, press the electrophoresis tank of claim 1, it is characterized in that the outside at gel mould, amberplex is set, thereby the space between the electrode is separated into the chamber that is parallel to each other, intermediate cavity is the separation chamber, two chambers adjacent with the separation chamber are developing room, and two chambers of close electrode are electrode chamber (the moon or anode chamber).
3, by the electrophoresis tank of claim 1 or 2, it is characterized in that described gel mould is 2～4, its thickness of every is 0.2～2 millimeter.
4, by the electrophoresis tank of claim 2, it is characterized in that the plasma exchange membrane is anion-exchange membrane and/or cation-exchange membrane.
5, by the electrophoresis tank of claim 1 or 2, it is characterized in that chamber is 3～5.
6, a kind of isoelectric point electrophoresis separating method for preparing type is characterized in that utilizing the electrophoresis tank of one of claim 1～4, and this method comprises the steps:
(1) sample liquid to be separated that the pH value is transferred to the isoelectric point of target protein feeds the separation chamber;
(2) the pH value is equaled target sample isoelectric points of proteins and electricity and lead little buffer solution feeding developing room;
(3) electrode solution is fed in the electrode chamber;
Loading constant DC between (4) two electrodes presses.
7,, it is characterized in that buffer solution is selected from a kind of of trishydroxymethylaminomethane-glycine, trishydroxymethylaminomethane-acetic acid, acetic acid-SAS by the electrophoresis separating method of claim 6.
8, by the electrophoresis separating method of claim 6, the flow velocity that it is characterized in that feeding the sample liquid to be separated of separation chamber is 100～1000ml/ branch.
9, by the electrophoresis separating method of claim 6, the flow velocity that it is characterized in that feeding the buffer solution of developing room is 200～2000ml/ branch.
10, by the electrophoresis separating method of claim 6, the concentration that it is characterized in that buffer solution is 0.005～0.1 mol.
11, by the electrophoresis separating method of claim 6, it is characterized in that putting on interelectrode voltage is 20～500 volts, and current strength is less than 100 milliamperes.
12, by the separation method of claim 6, it is characterized in that sample liquid is the liquid that contains BHb, bovine serum albumin.
13,, it is characterized in that sample liquid is that wherein interleukin-6 content is about 0.22 grams per milliliter through the broken liquid of the interleukin-6 inclusion body of rough segmentation by the electrophoresis separating method of claim 6.
14, by the electrophoresis separating method of claim 6, it is characterized in that sample liquid is urokinase half elaboration, it is 4000 unit of activity/milligram albumen than work.
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| CN95101184A CN1051245C (en) | 1995-01-20 | 1995-01-20 | Preparation type isoelectric point electrophoresis separating method and equipment |
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| WO1989000205A1 (en) * | 1987-07-02 | 1989-01-12 | American National Red Cross | New protein c activator |
| EP0323948A2 (en) * | 1988-01-05 | 1989-07-12 | Monsanto Company | Continuous isoelectric separation |
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| US6110434A (en) * | 1998-02-25 | 2000-08-29 | Alumitech Of Cleveland, Inc. | Recovery of products from non-metallic products derived from aluminum dross |
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| EP0287513A2 (en) * | 1987-04-11 | 1988-10-19 | Ciba-Geigy Ag | An isoelectric focusing process and a means for carrying out said process |
| WO1989000205A1 (en) * | 1987-07-02 | 1989-01-12 | American National Red Cross | New protein c activator |
| EP0323948A2 (en) * | 1988-01-05 | 1989-07-12 | Monsanto Company | Continuous isoelectric separation |
| CN1056261A (en) * | 1990-04-28 | 1991-11-20 | 曹成喜 | Multifunctional free-electrophoresis technology |
| US6110434A (en) * | 1998-02-25 | 2000-08-29 | Alumitech Of Cleveland, Inc. | Recovery of products from non-metallic products derived from aluminum dross |
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| CN100439916C (en) * | 2000-12-18 | 2008-12-03 | 贝克顿迪金森公司 | Electrophoresis device, electrophoreis method using electrophoresis device and utilization of electrophoresis device |
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| CN1127157A (en) | 1996-07-24 |
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