CN101328206A - Non-denaturing preparative electrophoresis - Google Patents
Non-denaturing preparative electrophoresis Download PDFInfo
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- CN101328206A CN101328206A CNA2008100202949A CN200810020294A CN101328206A CN 101328206 A CN101328206 A CN 101328206A CN A2008100202949 A CNA2008100202949 A CN A2008100202949A CN 200810020294 A CN200810020294 A CN 200810020294A CN 101328206 A CN101328206 A CN 101328206A
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Abstract
The invention relates to an electrophoretic separation device and a technology used for laboratorial and large-scale separated and purified biological samples. The invention mainly relates to the electrophoretic separation device which is a device capable of being used for performing the non-denaturing electrophoretic preparation technology, and a method required for completing the electrophoretic preparation technology by the device. The device mainly comprises a cooling chamber which is provided with a good heat exchange structure, a vertical electrophoretic plate with non-denaturing polyacrylamide gel arranged in the cooling chamber, a concentrating and collecting chamber which is used for sample collection, and an electrophoretic cell. The technology mainly comprises screening of collection time of purified proteins and a method for collecting the purified proteins. The device and the technology have the characteristics of large processing capacity, high resolution, convenient operation and so on, can be used for preparing laboratorial and large-scale biological products and biological medicines, and have considerable economic benefit and social benefit.
Description
Technical field
The present invention relates to a kind of novel electrophoretic separation device and technology thereof that is used for isolated protein, belong to technological field of biochemistry.
Technical background
1808, just the someone finds electrophoresis (EI electrophoresis) phenomenon, but the Tiselius of Sweden has set up " free electrophoresis " up to nineteen thirty-seven, successfully serum protein is divided into 5 main components, and electrophoretic technique is widely used in biochemical analysis.After this, weiland in 1984 etc. have developed again with the electrophoresis method of paper as upholder, make electrophoretic technique obtain further application and development.Through constantly making great efforts, particularly nearly 30 years development, electrophoretic technique has all obtained bigger perfect from theory into action, become indispensable method in protein, the foranalysis of nucleic acids
[1]
The kind of electrophoretic technique is a lot, but the two big classes that added up to, wherein a class is no support electrophoretic technique, another kind of is the support electrophoretic technique.The latter mainly includes paper electrophoresis, cellulose acetate membrane electrophoresis, glass fibre film electrophoresis, starch gel electrophoresis, agarose gel electrophoresis, counterimmunoelectrophoresis, rocket electrophoresis, polyacrylamide gel electrophoresis etc.
[2]Wherein, most widely used protein electrophorese be polyacrylamide gel electrophoresis (Polyacrylamdeoel Electrophoresis, PAGE).
Start from early sixties with polyacrylamide gel as the electrophoresis upholder.Polyacrylamide is by monomer acrylamide and comonomer N, and N-first justice bisacrylamide is crosslinked and the copolymerization product that forms is a kind of three-dimensional gel network.The concentration of acrylamide and methylene diacrylamide is depended in the aperture of this network, and the concentration of the two is high more, and the gel aperture that makes is just more little.Have many advantages with polyacrylamide gel as the electrophoresis upholder: be transparent in the finite concentration scope 1), physical strength is good, and is flexible, and chemical stability is good; 2) acrylamide does not have charged group, does not produce electric osmose during electrophoresis; 3) can control gel strength as required, make the gel in different apertures; 4) molecular sieve effect and electrocharge effect are combined, have very high resolving power; 5) amount of samples is few, and electrophoresis time is short, and equipment is simple
[2]
Electrophoresis also is a kind of effective analytical test instrument as the basic isolation technique of analysis and profiling protein matter complex mixture in the biological technical field undoubtedly.In proteinic Biochemical Research, to compare with other analysis mode separation means, electrophoretic separation technique has mild condition, resolving power height, easy to operate, advantage such as amount of samples is few.
Yet, for the SDS-polyacrylamide gel electrophoresis (SDS-Polyacrylamide Gel ElectroPhoresis is called for short SDS-PAGE) of routine.Owing in this electrophoresis system, adopt SDS (sodium laurylsulfonate) anionic detergent, when forming electronegative protein-SDS mixture, can destroy between the protein molecule and and other material molecule between non covalent bond, make protein denaturation and change its original conformation, therefore inevitably can influence the activity of target protein, can not be widely used in the proteic preparation separation.
Along with developing rapidly of biotechnology, at some important biological substance, the desired preparation separation and purification of various biologically active substances such as for example various special efficacy pharmaceutical grade proteins, hormone, be that people urgently expect the new problem that solves, also be to form pressing for of rising high tech, high profit industry.Present separation at these biologically active substances, main still be separated into the master with the post of routine, and these conventional posts separation also obviously exist disengaging time long, just seem very important of the loaded down with trivial details defective that waits some to overcome of separating step, the research of therefore carrying out to be used for the preparative electrophoresis of purpose sample separation purifying.
The electrophoretic form of preparation can be divided into three kinds according to separation principle at present
[3]: a kind ofly be called continuous wash-out gel electrophoresis.Be to utilize conventional polyacrylamide gel electrophoresis or agarose electrophoresis to separate.But also can only the isolated molecule quality differ have only 2% or iso-electric point differ the albumen of 0.1pH unit.
Second kind prepares electrophoresis is to utilize the isoelectrofocusing of carrier ampholyte to carry out separation and purification.As everyone knows, as amphotericeledrolyte protein, its carried charge changes with the variation of pH, when pH equaled certain proteic iso-electric point, this albumen just can not move in electric field.Vesterberg had synthesized carrier ampholyte (AmPholin in 1966, form by the isomer of the many carboxyls of many aliphatic polyaminos and homologue, molecular weight is generally less than 1000), thus aminoly obtain different iso-electric point compounds with the ratio of carboxyl and can form the pH gradient by changing in each molecule.Under electric field action, it can form the pH gradient within the specific limits.Isoelectric focusing electrophoresis designs according to this principle, and promptly it can make range protein separate by the difference of iso-electric point in pH gradient environment.Amphoteric substances such as protein can positively charged or negative electricity in the solution of different pH.If there is the pH field simultaneously on the direction of electric field, when the iso-electric point (pI) of particle just in time equals the pH value of the solution of its present position, particle just not with clean electric charge, can not move under electric field action yet.Even left the position that original pH-pI is ordered under this particle power effect outside (as thermodiffusion), under electric field action, can produce a kind of focussing force, be returned to original position.Utilization produces the character of " electrophoresis " and " focusing " under electric field action, developed the electrophoresis and the isoelectrofocusing of amphoteric substances such as high accuracy analysis protein, in order to separate and some enzyme of purifying, physiologically active substance.The upholder of isoelectric focusing electrophoresis has polyacrylamide gel, sepharose and sucrose solution etc.Sucrose solution is mainly used in the isoelectrofocusing of preparation type as upholder.Isoelectrofocusing has many original advantages as a kind of effective preparation method, and is constant substantially as the physico-chemical property and the biological property of sample, because focusing effect can play inspissated to original very rare sample.
Isoelectrofocusing is used widely at everyways such as scientific research, clinical medicine, agricultural and food analyses, is the important means of measuring isoelectric points of proteins.And as a vital technology, itself is also in continuous development.As a kind of method of isolated protein, its resolving power will be higher than first kind of above-mentioned mode relatively, and the albumen unchangeability.The column isoelectrofocusing of the LKB company that relatively is typically Sweden the earliest prepares electrophoresis apparatus.It uses sucrose density gradient as supporting dielectric, forms the pH gradient with carrier ampholyte.But operate tediously longly, loaded down with trivial details, now be eliminated.
The third is to utilize solid phase pH gradient isoelectric focusing electrophoresis to separate.This is the highest a kind of electrophoresis separating method of present resolving power, by being similar to concentration gradient preparing gel method, control acid, alkali amphotericeledrolyte ratio just can prepare the gel of Stationary pH gradient.Because in the gel polymerisation process, as amphotericeledrolyte acrylamide derivative can be fixed in the gel with the polymerization of acrylic amine covalency.Form fixed pH linear gradient.This has just guaranteed that in the focusing process pH gradient can not change because of the influence of other factors, thereby has further improved the scope of application and the resolving power of isoelectrofocusing.The Iso Prime of U.S. Hoefer company release in 1993 utilizes this principle to separate.Fractional dose can be from 100 μ g-1g, and resolving power can reach 0.01pH.
Although now had electrophoresis method can be used for the preparation of a small amount of albumen or sample, but, regrettably, up to now electrophoretic separation technique is applied to the laboratory in a small amount and the production of scale protein example when preparing, no matter be its separation accuracy or its practical degree, all far away can not be fully up to expectations (fail to reach be used for the such level of protein analysis), because these electrophoresis methods are difficult to realize serialization production in batches, apparatus and method are more complicated all, and with high costs, limited the application of this kind electrophoretic technique in protein separation greatly.Therefore study a kind of have easy, practicality, the preparative electrophoresis separation method just seems particularly important accurately.The present invention's novel electrophoretic separation technique of research and utilization by experiment replaces conventional art to realize separation and purification biological and biochemical substances, not only can reduce production costs significantly, and will have a tremendous social and economic benefits.
Summary of the invention
The objective of the invention is to design a kind of high and preparative electrophoresis separating device applied widely of separation accuracy that has, be applied to be used for gene engineering product in experiment and the scale production, medicinal organism goods etc. have the final purifying of the biological products of important value.
Content of the present invention comprises: a kind of polyacrylamide gel vertical electrophoresis device and using method thereof of non-sex change, this device is by the separate part of circulating condensing device, be used to collect the concentrated and purified chamber of sample, and the tiselius apparatus that connects by semi-permeable membranes constitutes, by the collection of indication electrophoresis showed target protein, can be used for laboratory electrophoresis preparation small-sized and scale separation and purification biologically active substance and separate.
The isolating principle of the present invention: the electrophoresis technology of preparing of a kind of non-sex change that invention provides, separate preparation according to biological sample bulk of molecule and electrically charged two factors, when electrically charged and with different size and shape biological sample passes through the polyacrylamide gel of certain pore size, the degree that blocked is different and show different mobilities, by the electrophoresis track of indication B plate electrophoresis indication purpose sample, the collection of A plate is finished amphiprotic substance and is carried out separation and purification and analysis.
The object of the present invention is achieved like this: the polyacrylamide gel that adds non-sex change in the vertical electrophoresis chamber of placing, in the B plate, add specific staining reagent, this staining reagent can be realized non-specific the combination with albumen, the electrophoresis track that can show target protein in electrophoresis, the collection of target protein is finished according to proteic swimming in the B plate in the A plate.Because A, do not contain SDS in B two plates, therefore prepared albumen can loss of activity, and the separate part that includes the circulating condensing device that this device comprised, and can prevent the protein denaturation inactivation that causes owing to heat production in electrophoresis.In addition, contain the concentrated and purified chamber of collecting sample in the device, can guarantee being communicated with of collecting tank and tiselius apparatus owing to contain semi-permeable membranes, guarantee electrophoreticly normally to carry out, less collecting tank can guarantee the concentrated of sample.This device can concentrate rarer biological sample, concentrates the back dialysis.This technology is not only applicable to the biological sample scale of carrying out is separated and purifying, and is applicable to the separation and purification and the analysis of a small amount of or micro-example.
The indication electrophoresis that is used for the B plate among the present invention can be to adopt to finish indication by pigment with combining of target protein, but be not limited to the indication of using pigment.The indication electrophoresis that is used for the B plate among the present invention, can be resolving power higher in conjunction with technology such as isotopic labelings, also can be to finish collection by drawing sample swimming curve.Proteinic basic chemical structure unit is an amino acid, and protein is as a kind of amphotericeledrolyte, itself have certain positive charge, thereby to contain anionic dyestuffs such as sulfonic group or dye matrix be that the metallized dye of negative ion just can be by electrostatic attraction and intermolecular Van der Waals force and protein bound
[4-6], this also is that triarylmethane compound dyestuff Xylene Brilliant Cyanine G G-250 measures in the ultimate principle of method of protein and the SDS-PAGE electrophoresis and utilizes bromjophenol blue to carry out the theoretical foundation of forward position indication.The present invention utilizes pigment to combine the indication that forms in the electrophoresis process with the non-selectivity of target protein just, instruct the collection of target protein, for can't bonded albumen selecting by finishing collection in conjunction with technology such as isotopic labeling or by drawing sample swimming curve.
Description of drawings
Fig. 1 is non-sex change preparative electrophoresis separation method schematic diagram.
1,2 is respectively the groove tiselius apparatus among the figure, and 5,6 is target protein, and 7,8 is the foreign protein of mobility speed greater than target protein, and 3,4 is the foreign protein of mobility speed less than target protein.9,10 gel glass spacer plates, 12 is semi-permeable membranes, and 11 for concentrating collecting chamber, and 13,14 are following groove tiselius apparatus.
Fig. 2 is the vertical view of this device.
1 is last groove tiselius apparatus among the figure, and 2 are following groove tiselius apparatus, and 3 is electrode jack, and 4 is the prolong water outlet
Fig. 3 is the front view of this device.
Wherein Fig. 3 a is the right side view of this device, and Fig. 3 b is the left side view of this device.
1 is the application of sample comb among Fig. 3 a, and 2 is the water of condensation water outlet, and 3 is the electrophoresis sheet glass, and 4 are following groove tiselius apparatus, 5 anode electrode jacks, and 6 is the sample collection groove, 7 is the negative electrode plug wire hole among Fig. 3 b, 8 grooves, 9 is the water of condensation water inlet.
Fig. 4 is the condensing works figure of this device.
Wherein Fig. 4 a is the left side view of condensing works, and Fig. 4 b is the right side view of condensing works.
1 is the water of condensation water inlet among Fig. 4 a, and 2 is the water of condensation water outlet among Fig. 4 b
Fig. 5 is the film wiring layout of the concentrated collecting chamber of this device.
Fig. 5 a wherein, Fig. 5 c, Fig. 5 e is respectively the semi-permeable membranes support and overlooks, and faces and lateral plan; Fig. 5 b, 5d, 5f is respectively collecting chamber and overlooks, and faces and lateral plan.
Fig. 6 is the synoptic diagram of this device application of sample comb.
Fig. 6 a wherein, Fig. 6 b, Fig. 6 c are respectively overlooking of this device application of sample comb, face and lateral plan.
Fig. 7 is the electrophoresis plate synoptic diagram of this device.
Fig. 7 a wherein, Fig. 7 b are respectively the synoptic diagram of electrophoresis plate and following electrophoresis plate.
Fig. 8 is the pictorial diagram of this device.
Wherein Fig. 8 a is a front view in kind, and Fig. 8 b is a lateral plan in kind, and Fig. 8 c is a gel making device, and Fig. 8 d is the sample collection groove.
Fig. 9 is protein comparison of electrophoresis behavior in non-sex change electrophoresis after using indication damping fluid and sample pipetting volume damping fluid of differing molecular size.
Fig. 9 a wherein, Fig. 9 b is respectively recombination human serum albumin thymic peptide fusion protein as one (69kd), electrophoretic migration figure under indication sample-loading buffer and sample sample-loading buffer in non-sex change electrophoresis, Fig. 9 a is the collection of illustrative plates that fusion rotein begins swimming, and Fig. 9 b is the collection of illustrative plates of fusion rotein swimming during near the bottom.
Fig. 9 c wherein, Fig. 9 d is respectively reorganization asparaginase (144kd) electrophoretic migration figure in non-sex change electrophoresis under indication sample-loading buffer and sample sample-loading buffer, Fig. 9 c is the swimming collection of illustrative plates of target protein (sample buffer) and standard protein (indication damping fluid), and Fig. 9 d is the swimming collection of illustrative plates of target protein under indication sample-loading buffer and sample pipetting volume damping fluid.
Wherein Fig. 9 e is thymus gland 28 peptides (3.2kd) electrophoretic migration collection of illustrative plates in non-sex change electrophoresis under indication sample-loading buffer and sample sample-loading buffer.
Wherein Fig. 9 f is recombination human serum albumin thymic peptide fusion protein as one (69kd) electrophoretic migration icones in non-sex change electrophoresis under the indication sample-loading buffer.
Figure 10 is the SDS-PAGE of the non-sex change electrophoresis purification of Recombinant human serum albumin thymic peptide fusion protein as one of preparation property
Wherein swimming lane 1, mark albumen, and 2, recombination human serum albumin thymic peptide fusion protein as one sample, 5,6 purpose fusion roteins for collection, 3,4,7,8,9,10,11 is foreign protein.
Figure 11 is the determination of activity of recombination human serum albumin thymic peptide fusion protein as one.
Figure 12 is the SDS-PAGE of the non-sex change electrophoresis purification of Recombinant Asparaginase of preparation property
The present invention is described in detail and illustrate below in conjunction with this device accompanying drawing.
With reference to the non-sex change preparative electrophoresis of accompanying drawing 1-separation method schematic diagram. Its separation process of the present invention is: add among the A of separation chamber The sample liquid that does not contain the dyestuff sample-loading buffer, the B of separation chamber passes into and adds the sample liquid that contains the dyestuff sample-loading buffer, two electrodes Between load constant DC voltage. A plate electrophoresis target protein molecule under such condition (5, in the accompanying drawing 1 with little black Piece ■ represents) from different foreign protein (3,7, in the accompanying drawing 1 with triangle ▲ and little black circle ● expression) inevitable under electric field action Electrophoretic migration takes place; The target protein molecule that the B plate contains (6, in the accompanying drawing 1 with black stripe
Expression) from different foreign protein (4,8, in the accompanying drawing 1 with positive ash side bar
Tiltedly grey side's bar represents
Expression) electricity takes place under electric field action The swimming migration also presents a colour band, and certain density polyacrylamide gel is passed respectively in the swimming under electric field of A plate destination protein Enter the collecting chamber (11) with inspissation, the collection of A plate destination protein is finished collection according to the color indication of B plate, thereby Being separated from each other of realize target protein.
Top view with reference to this device of accompanying drawing 2-. As seen going up groove (1) is the container of splendid attire anode buffer liquid, when electrophoresis by two Piece glass electrophoresis plate forms the container of splendid attire buffer solution, is the bottom that electrode hole (3) and inert electrode are distributed in upper groove at its left. Lower groove (2) is splendid attire negative electrode buffer solution, and there is inert electrode in bottom branch, and right-hand is condenser pipe delivery port (4).
Front view with reference to this device of accompanying drawing 3-. (9) and left side below (2) is condensation-water drain above the right side of device. Application of sample comb (1) is arranged above device, be used to form loading slot. There are two electrophoresis glass plates (3) centre at device, comprises Certain density polyacrylamide is used for finishing electrophoretic separation. Below device, be useful in the lower groove (4) and concentrate the collection of collecting Groove (6), feeder are attached to groove (8) vertically in lower groove; Installing right-hand bottom is negative electrode plug wire hole (5,7).
Condensing unit figure with reference to this device of accompanying drawing 4-. (2) and left side below (1) is condensed water above this device right side Water in-out port prevents sample in electrophoresis because heating produces sex change.
Film installation diagram with reference to the concentrated collecting chamber of this device of accompanying drawing 5-. Penetrating film can be fixed on the support during assembling and inlay In collecting chamber the place ahead, collecting chamber leaves grid, guarantees that collecting chamber communicates with lower groove buffer solution by penetrating film, finishes electrophoresis. Receive Collect indoor rear wall and be close to the electrophoresis glass plate, as far as possible little space guarantees that the sample of electrophoretic separation can not be diluted.
Advantage of the present invention:
1 is simple in structure, and stable performance is with low cost.Draw materials conveniently, protein Preparation adopts inexpensive plain polypropylene acrylamide gel electrophoresis.
2 need not cut glue and secondarily purified again, and target protein is directly collected acquisition at collecting chamber.
3 as far as possible little receiving tanks, maximum collected volume only is 2.0ml, makes that separating target protein need not concentrate.
4 receiving tanks are connected with following groove electrode buffer by semi-permeable membranes, finish proteic electrophoresis swimming process, and big as far as possible penetrating area makes that electrode buffer and various ion can be unobstructed.
5 condensing chambers have guaranteed favorable cooling effect, guarantee that the purpose sample thermally denature can not occur and cause loss of activity.
Each parts of 6 electrophoresis chambers are installed simple, and gel is convenient to remove, are convenient to clean, dismantle and maintenance.
Embodiment
Put to the proof explanation below in conjunction with specific embodiment.
The big small protein of embodiment 1 differing molecular adds the investigation of pigment indication rear electrophoresis behavior
1 experimental design purpose: the non-sex change electrophoresis of the present invention is by adding the electrophoresis behavior of the sample-loading buffer indication target protein that contains the Coomassie brilliant blue dyestuff in the sample, it is the electrophoresis position that the B plate shows target protein, the A plate is collected target protein according to the indication of B plate, can but contain the electrophoretic migratory behaviour that the sample-loading buffer of examining the bright blue dyestuff of Ma Shi change sample protein? contain with the electrophoretic migration that does not contain the upper boom damping fluid target protein of examining the bright blue dyestuff of Ma Shi and change? The effects the protein molecular of different sizes, thymus gland 28 peptides (3.2kd), recombination human serum albumin thymic peptide fusion protein as one (69kd), reorganization asparaginase (144kd) adds and contains and do not contain the sample-loading buffer of examining the bright blue dyestuff of Ma Shi, electrophoresis dying is investigated the two electrophoresis behavior.
The pre-treatment of 2 samples:
1) processing of recombination human serum albumin thymic peptide fusion protein as one fermented sample:
Behind centrifugal 15 minutes of the pichia spp fermented liquid 8000r/min, getting supernatant, to record protein content be 1.2g/L, sample thief ultrafiltration, dialysis, the desalination of G-25 gel.
2) processing of reorganization asparaginase fermented sample:
Fermented liquid is centrifugal, removes supernatant, and wet thallus is suspended in the shell-broken liquid (5mmol/LEDTA, the 150mg/L N,O-Diacetylmuramidase, pH 7.0 for w/v, 35% sucrose) of 5 times of volumes, and 37 ℃ are stirred 35min.Slowly add 1M MnCl
2, precipitate nucleic acids and bacterial chip, ice-water bath stir behind the 1h in 4 ℃ of 8000rpm frozen centrifugation 15min, abandon precipitate crude enzyme liquid, add 60% (NH
4)
2SO
4The back is centrifugal, and supernatant adds 90% (NH
4)
2SO
4, centrifugal back collecting precipitation, precipitation is dissolved with appropriate amount of buffer solution, the solution ultrafiltration after the dissolving, dialysis, the desalination of G-25 gel.
3) thymus gland 28 peptides:
Thymus gland 28 peptides of chemosynthesis.
2 deposition conditions
1) electrophoretic buffer: glycine-Tris damping fluid, reference literature [7] preparation.Specifically be formulated as follows:
Tris-glycine electrophoretic buffer.This damping fluid contains 25mmol/L Tris alkali, 250mmol/L glycine, and (the electrophoresis level pH8.3), but does not contain SDS, and it is standby to be made into the 5x stock solution.Dissolving 15.1gTris alkali and 94g glycine in the 900ml deionized water mended to the 1000ml amount with deionized water and then to be become the 5x stock solution.
2) non-sex change sample-loading buffer:
A: be used for the sample loading buffer of sample collection, that is: 5 * non-sex change does not contain and examines the bright orchid of Ma Shi and the sds gel sample loading buffer is made up of following:
50mol/L?Tris.Cl(pH?6.8)
50% glycerine
The native gel sample loading buffer that does not contain dithiothreitol (DTT) (DTT) or SDS can be stored in room temperature.
B: the sample-loading buffer that is used to indicate, that is: 5 * non-sex change does not contain the sds gel sample loading buffer and is made up of following:
50m?mol/L?Tris.Cl(pH?6.8)
0.1% examines the bright orchid of Ma Shi
10% glycerine
The native gel sample loading buffer that does not contain dithiothreitol (DTT) (DTT) or SDS can be stored in room temperature.
3 operating process reference literatures
[7]Carry out, that is: the sample of equivalent adds A respectively, the B sample-loading buffer, but need not to boil, directly application of sample electrophoresis behind the centrifugal 10min of 8000r/min dyes, decolouring.
4 results:
See protein comparison of electrophoresis behavior in non-sex change electrophoresis after using indication damping fluid and sample pipetting volume damping fluid of Fig. 9-differing molecular size.
From Fig. 9 a-Fig. 9 e protein of differing molecular size as can be known, thymus gland 28 peptides (3.2kd), recombination human serum albumin thymic peptide fusion protein as one (69kd), the reorganization asparaginase (144kd) its under non-sex change deposition condition, the behavior of the electrophoresis swimming under its indication sample-loading buffer and the sample sample-loading buffer is approaching substantially, this result also points out, and can indicate the collection of sample protein in non-sex change electrophoresis with examining the bright orchid of Ma Shi.And from Fig. 9 f as can be known, sample is in the indication sample-loading buffer, and its electrophoresis swimming is high-visible, can not disappear in whole swimming process, can be used for showing the collection of sample protein at non-sex change electrophoresis.
Embodiment 2: separate the non-sex change electrophoresis purifying and the determination of activity of pichia spp fermentation recombination human serum albumin thymic peptide fusion protein as one sample with non-sex change preparative electrophoresis
The pre-treatment of 1 sample:
With embodiment 1
2 deposition conditions
1 electrophoretic buffer
With embodiment 1
2 non-sex change sample-loading buffers:
With embodiment 1
3 electrophoresis operating processes, reference literature
[7]Carry out, that is: the sample of equivalent adds A respectively, the B sample-loading buffer, but need not to boil application of sample electrophoresis behind the direct centrifugal 10min of 8000r/min, electrode current wherein: concentrate glue constant current 15mA, separation gel constant current 30mA, when indicating the purpose fusion rotein soon near the receiving tank bottom, every interval 15 minutes, collect sample, swum until the indication sample.
4 electrophoresis purification result:
See the SDS-PAGE of the non-sex change electrophoresis of Figure 10-preparation property purification of Recombinant human serum albumin thymic peptide fusion protein as one.
It is 95% recombinant protein that the result obtains purity, confirms that this kind device can be used for protein purification.
The activation analysis of 5 purification of samples:
The activation analysis of reorganization target protein is according to document
[8]Carry out: the fresh anticoagulant heparin venous blood of health adult, an amount of Hanks liquid dilution adds in the centrifuge tube of the lymphocyte separation medium that has had 1/3 amount of filtrate, 20000 left the heart 20 minutes, sucking-off contains lymphocytic muddy band puts in the centrifuge tube, adds an amount of Hank ' s liquid washing, shakes up, leave heart 3-5 minute with per minute 1500, abandoning supernatant, wash 2 times after, in throw out, add an amount of Hank ' s of people liquid, mixing, 30 minutes (once) of 45 ' C water bath with thermostatic control insulation every jolting in 5 minutes.Use Hank ' s liquid to be made into and transfer to 5 * 10
5/ ml cell concn.Get 0.2ml put into small test tube with different concns medicine to be measured with Hank ' s liquid dilution mixing, put in 37 ℃ of every pipes of water-bath 1h each add prepare add 0.2ml sheep red blood cell (SRBC) suspension (concentration be take off E acceptor thymus gland T concentration of cell suspension 8-10 doubly, be about 10
8Ten thousand/ml) shake up 500r/min gently, centrifugal 3min, 4 ℃ of placements are spent the night, and fix Giemsa staining with glutaraldehyde.
The result: oily mirror is observed down, and each 3 of lymphocyte adhesion or 3 above sheep red blood cell (SRBC) persons are the E garland, calculate the percentage ratio of rosette forming cell (RFC) in 200 lymphocytes.See the determination of activity of accompanying drawing 11-recombination human serum albumin thymic peptide fusion protein as one.
Results suggest utilizes the fusion protein sample of electrophoresis apparatus purifying of the present invention to show the biological activity similar to synthetic Zadaxin.
Embodiment 3: with the non-sex change electrophoresis purifying and the determination of activity of non-sex change preparative electrophoresis separating Escherichia coli fermentation reorganization Asparaginase sample
The pre-treatment of 1 sample:
With embodiment 1
2 deposition conditions
1 electrophoretic buffer
With embodiment 1
2 non-sex change sample-loading buffers:
With embodiment 1
3 electrophoresis operating processes, the operating process reference literature
[7]Carry out, that is: the sample of equivalent adds A respectively, the B sample-loading buffer, but need not to boil application of sample electrophoresis behind the direct centrifugal 10min of 8000r/min, electrode current wherein: concentrate glue constant current 15mA, separation gel constant current 30mA, when indicating the purpose fusion rotein soon near the receiving tank bottom, every interval 15 minutes, collect sample, swum until the indication sample.
4 electrophoresis purification result:
See the SDS-PAGE of the non-sex change electrophoresis of accompanying drawing 12-preparation property purification of Recombinant Asparaginase.
It is 95% recombinant protein that the result obtains purity, confirms that this kind device can be used for protein purification.
The enzyme activity determination of 5 purification of samples and result
Get 1ml pH8.4 borate buffer and 0.04mol/L altheine substrate respectively and add mixing in vitro, 37 ℃ of preheating 5min add 5～20 μ l testing samples then, and 37 ℃ accurately add 1ml 50% trichoroacetic acid(TCA) stopped reaction behind the reaction 15min.Draw the above-mentioned reaction solution of 0.5ml to 3.5ml distilled water, add the 1ml Nessler's reagent again, room temperature leaves standstill 15min, 500nm colorimetric on 721 spectrophotometers.
The result: the enzyme work of crude enzyme liquid is: 342.77; Utilize the sample enzyme behind apparatus of the present invention purifying to live: 338.33, the sample of this presentation of results behind electrophoresis apparatus purifying of the present invention shown the enzymic activity similar to former state.
Reference:
[1] multifunctional free-electrophoresis technology publication number: CN 1056261A
[2] Cai Shiyingren will is built electrophoretic principle, application and progress chromatogram 1992,10 (4), 207-210
[3] protein electrophorese experimental technique (second edition) monarch Guo Yao Science Press 2005
[4] Luan Jimei, Hu Mingjin, the progress Anhui College of Education journal 2004,22 (3) of Zhang Xiaodong organic dye spectrophotometric protein determination: 60-63
[5]Tal?M，Silberstein?A，Nusser?E.Why?does?Coomassie?Brilliant?Blue?R?interactdifferently?with?different?proteins？A?partial?answer.[J].J?Bio?Chem，1985，260(18)：9976-9980
[6]Suzuki?Y.Guidance?for?Selecting?the?Measurement?Conditions?in?the?Dye?bindingMethod?for?Determining?Serum?Protein：Theoretical?Analysis?Based?on?the?ChemicalEquilibrium?of?Protein?Error[J].A?analyt?ical?Sciences，2001，17(11)：1263～1268.
[7] molecular cloning experiment guide (second edition) Sa Mubulu work, Jin Dongyan, Li Mengfeng translate Science Press 1990
[8] the Chinese drug standard (WS ,-XG-042-2000-2003) 2003 the 4th the volume the 6th phase (total 338), 17-18
[9] Liu Jingjing, Jinjian duty, Wu Wutong etc. the extraction of bacillus coli L-asparaginase enzyme and purifying [J]. medicine biotechnology, 1995,2 (1): 16-19
Claims (4)
1. the electrophoretic separation device of a preparation property comprises the required method of this device that realizes.Described electrophoretic separation device is to utilize preparative electrophoresis device of the present invention can biomacromolecule especially be fit to multiple mixed protein and carry out separation and purification, and can realize that proteic separation reaches spissated purpose to difference.
2. the described preparative electrophoresis device of claim 1 has following feature: a kind of vertical electrophoresis device that the polyacrylamide gel of non-sex change is housed, the electrophoretic separation that can be used for the small-sized and scale separation and purification biomacromolecule material in laboratory, this device includes the cooling room of circulating condensing device, be used to collect the concentrated and purified chamber of sample, and the tiselius apparatus that connects by semi-permeable membranes constitutes.
3. in the described preparative electrophoresis device of claim 2, dress is not contain the non-denaturing polyacrylamide gel of SDS in the vertical electrophoresis plate, and by A, two formations of B can be used for the collection of purpose sample, and the purpose sample activity of collecting is unaffected.
4. in the described preparative electrophoresis device of claim 2, wherein the A plate is used for the preparation of target protein, finishes the concentrated collection of purpose sample.
In the described preparative electrophoresis device of 5 claims 2, wherein the B plate can be used for the collection indication of target protein, and after the condition in later stage was determined, B also can be used for the concentrated collection of above-mentioned target protein.The indication that the B plate is used for the purpose sample can be to utilize pigment to indicate with combining of purpose sample, also can be resolving power higher in conjunction with technology such as isotopic labelings, also can be to finish collection by drawing sample swimming curve.
In the described preparative electrophoresis device of 6 claims 2, it is characterized in that whole electrophoresis apparatus can be designed to have the electrophoresis apparatus of cooling system.
In the described preparative electrophoresis device of 7 claims 2, be provided with the concentrated and purified chamber that has the semi-permeable membranes formation.Described concentrated and purified chamber is characterized in that realizing connecting by the semi-permeable membranes of certain pore size between concentrated and purified chamber and the following groove tiselius apparatus, and the target protein or the peptide material that separate purification or concentrated dialysis do not had logical luring property.And small ion materials such as small molecules or electrophoretic buffer are freely passed through.
The required technology of the described preparative electrophoresis device of 8 claims 1 comprises, is used to prepare indicator and the collection method that target protein adopted that target protein shows.
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| CNA2008100202949A CN101328206A (en) | 2008-02-29 | 2008-02-29 | Non-denaturing preparative electrophoresis |
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| CNA2008100202949A CN101328206A (en) | 2008-02-29 | 2008-02-29 | Non-denaturing preparative electrophoresis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899425A (en) * | 2010-06-03 | 2010-12-01 | 中国海洋大学 | Method for separating and purifying scallop phenol oxidase |
| CN104792851A (en) * | 2015-04-20 | 2015-07-22 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Micropreparation-type gel electrophoresis device and use method thereof |
| RU214011U1 (en) * | 2022-03-28 | 2022-10-07 | Федеральное государственное бюджетное учреждение науки Федеральный исследовательский центр Тюменский научный центр Сибирского отделения Российской академии наук | Vertical prolamin electrophoresis device with external cooling circuit |
-
2008
- 2008-02-29 CN CNA2008100202949A patent/CN101328206A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899425A (en) * | 2010-06-03 | 2010-12-01 | 中国海洋大学 | Method for separating and purifying scallop phenol oxidase |
| CN101899425B (en) * | 2010-06-03 | 2012-07-11 | 中国海洋大学 | Method for separating and purifying scallop phenol oxidase |
| CN104792851A (en) * | 2015-04-20 | 2015-07-22 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Micropreparation-type gel electrophoresis device and use method thereof |
| CN104792851B (en) * | 2015-04-20 | 2017-04-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Micropreparation-type gel electrophoresis device and use method thereof |
| RU214011U1 (en) * | 2022-03-28 | 2022-10-07 | Федеральное государственное бюджетное учреждение науки Федеральный исследовательский центр Тюменский научный центр Сибирского отделения Российской академии наук | Vertical prolamin electrophoresis device with external cooling circuit |
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Open date: 20081224 |