CN104974243A - Beta-amyloid 1-42 oligomer as well as preparation method and identification method therefor - Google Patents
Beta-amyloid 1-42 oligomer as well as preparation method and identification method therefor Download PDFInfo
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Abstract
The invention discloses a beta-amyloid 1-42 oligomer. The beta-amyloid 1-42 oligomer is a mixture of oligomers and monomers mainly; the oligomers are identified through a Western blot method: the molecular weight is 12-20 KD, and the oligomers are tripolymers and tetramers; and the monomers are identified through the Western blot method: the molecular weight is 4-4.5 KD. The invention also discloses a preparation method and an identification method of the beta-amyloid 1-42 oligomer. The beta-amyloid 1-42 oligomer provided by the invention is relatively single in composition and relatively stable in product, and a good foundation is provided for establishing ideal and reliable AD models; the preparation method is simple, high in yield and time-saving; and the adopted SDS-PAGE in combination with the Western blot method has the advantages of high sensitivity, high specificity, convenience, high speed, simple operation, relatively low cost and the like.
Description
Technical field
The present invention relates to amyloid-beta field, be specifically related to a kind of A β 1-42 oligomer and preparation thereof, authentication method.
Background technology
Alzheimer's disease (Alzheimer ' S disease, AD) also referred to as senile dementia, be the brain degenerative disease of a kind of chronic, persistence, the degenerative occurring in senium or presenium, its clinical manifestation is hypomnesis, cognitive disorder, personality change etc.There is the pathology such as senile plaque, cellular neurofibrillary entanglement being core with amyloid-beta (beta-amyloid, A β) in the extracellular that the pathological characters of this disease mainly comprises pallium and hippocampus.The fourth-largest dead killer being only second to cardiovascular diseases, cancer and cerebral apoplexy at U.S. AD, current whole world AD number of the infected is up to 2,500 ten thousand according to statistics, China AD patient has reached more than 600 ten thousand, occupy first place in the world, the AD course of disease time is grown and there is no the medicine of thoroughly radical cure at present, the health of the mankind in its serious threat, brings tremendous influence to patient and family members.Therefore set up desirable and AD animal model reliably, the cause of disease of research and exploitation AD, pathologic process and searching and screening active drug are seemed particularly important.
A β be by amyloid precursor protein produce through the proteolyzing of β-and gamma-secretase containing 39 ~ 43 amino acid whose polypeptide, a large amount of depositions of A β cause the unit's sex change of senile plaque peripheral nerve and main causes of death in patient's AD brain.A β has multiple existence form in brain, as: lower molecular weight A beta monomers, A beta oligomers, insoluble A beta body etc.A beta monomers is the lower-molecular substance of solubility, but when born of the same parents' environment, as A beta monomers rapid aggregation can be brought out when pH, temperature, ionic concn, protein concentration etc. change, form the A beta oligomers of dimer, tripolymer or solubility, the less stable of A beta oligomers, easily transforms to A beta body.The kind of A β is divided into A β 1-39, A β 1-40, A β 1-41, A β 1-42 and A β 1-43, and wherein the toxicity of A β 1-42 is maximum, is the main pathogenic of AD, and therefore, research A β 1-42 is current focus.
In recent years, have scientist to propose, the relation that A beta body and memory impairment and neurocyte lack is little, infers that solubility A beta monomers and A beta oligomers are the main pathogenics of AD.The oligomer of domestic and international preparation, synthesis A β 1-42 is all unstable, and 4 amyloid is easily randomly formed various high molecular or low-molecular-weight mix products in vitro, and the inhomogenous mixture of this component directly has influence on accuracy and the reliability of experimental result.In recent years, many investigators prepare A β 1-42 oligomer by multiple method, but yield is all lower, unstable products, complicated component.The unstable products of A β 1-42 oligomer, complicated component, just cannot ensure the successful foundation of its guidance model, thus directly has influence on accuracy and the reliability of experimental result.Therefore, the A β 1-42 oligomer that prepared composition is more single, stable, the successful foundation that can be AD animal model is laid a good foundation, also for the pathogenesis of AD, pathologic process, searching and screening active drug provide effective foundation.
Summary of the invention
For deficiencies such as existing preparation, the oligomer instability of synthesis A β 1-42, complicated component, yield are low, the invention provides a kind of A β 1-42 oligomer of new synthesis.A β 1-42 oligomer composition provided by the invention is more single, product stable, provides good basis for setting up desirable and reliable AD model; Its preparation method is simple, yield is high, save time; The SDS-PAGE associated proteins immunoblotting analysis authentication method adopted, has the advantages such as sensitivity is high, high specificity, fast and easy, simple to operate, expense is lower.
The present invention is achieved by the following technical solutions:
A kind of A β 1-42 oligomer, described A β 1-42 oligomer is mainly the mixture of oligomer and monomer; Described oligomer is through Western blot electroresis appraisal: molecular weight is 12-20KD, is tripolymer and the tetramer; Described monomer is through Western blot electroresis appraisal: molecular weight is 4-4.5KD.
The above A β 1-42 oligomer, the preparation method of described A β 1-42 oligomer is: get A β 1-42 synthetic peptide, be at room temperature dissolved completely in HFIP, gained solution is transferred in centrifuge tube, with dry bath Nitrogen evaporator, HFIP is thoroughly dried up; Adding DMSO makes the solid bottom centrifuge tube dissolve completely, adds HEPES damping fluid, mixes, to obtain final product.
The preparation method of the above A β 1-42 oligomer, comprises the following steps:
1. by the A β 1-42 synthetic peptide of 1mg, add in 220-225 μ L HFIP, at room temperature leave standstill 30-60 minute, obtain consoluet solution;
2. the solution that step 1 obtains is transferred to the centrifuge tube of silication, at dry bath Nitrogen evaporator blowing up 8-15 minute, HFIP is thoroughly dried up, obtain transparent sheet-like solid and be sunken at the bottom of pipe;
3. in described centrifuge tube, add 40 μ l DMSO, the solid bottom centrifuge tube dissolved completely, then add pH value be 7.4 concentration be l0mM HEPES buffered soln, make the last final volume of solution be 1ml, mix, to obtain final product.
The authentication method of the above A β 1-42 oligomer, comprises the following steps:
1.SDS-polyacrylamide gel electrophoresis: get A β 1-42 oligomer solution 2 μ L, mix with 18 μ L 4X electrophoresis sample-loading buffers, then loading, electrophoresis applied sample amount is 5ul/ swimming lane, the glue of electrophoresis is the gradient glue of 4 – 12%, add at a swimming lane pre-dyed molecular weight standards that 10 μ L molecular weight are 3.6-260KD simultaneously, during electrophoresis, select 80V electrophoresis 30-40 minute, then turn 100v voltage and continue electrophoresis 2 hours;
2. transferring film: the protein band that electrophoresis is good is transferred on pvdf membrane, semidrying transferring film, the time is 6 minutes, by the pvdf membrane that takes a turn for the better by the size cutting of application of sample swimming lane, is then dipped into 5-10 minute in TBST solution by having cut out pvdf membrane;
3. close: be immersed in by pvdf membrane in the TBST solution containing 5% skim-milk, shaking table vibrates, shaking table vibrates at a slow speed, rotating speed can be 65-75 rev/min again, under room temperature condition, close 1 hour;
4. wash film: pvdf membrane step 3 obtained is immersed in TBST solution, and shaking table shakes rinsing 2-3 time, each 5 minutes;
5. primary antibodie is hatched: pvdf membrane step 4 obtained is immersed in the primary antibodie solution containing 6E10, incubated at room temperature 1 hour;
6. wash primary antibodie film: be immersed in by the pvdf membrane of hatching through primary antibodie in TBST solution, shaking table shakes rinsing 6 times, each 5 minutes;
7. two anti-to hatch: the pvdf membrane that rinsing is good is immersed in the two anti-solution containing horseradish peroxidase-labeled, incubated at room temperature 1 hour;
8. wash two anti-films: by being immersed in TBST solution through two anti-pvdf membranes of hatching, shaking table shakes rinsing 6 times, each 5 minutes;
9. chemoluminescence exposure: be evenly added in by HRP substrate solution on the pvdf membrane that step 8 is rinsed, carries out darkroom exposure imaging respectively within 5-30 second, therefrom select band clearly film as end-result.
Compared with prior art, the beneficial effect that the present invention obtains is:
1. A β 1-42 oligomer composition provided by the invention is more single, main containing 12-20KD oligomer, and purity more than 80% is another containing a small amount of 4-4.5KD monomer; A β 1-42 oligomer is very stable, and under 4 DEG C to room temperature condition, its molecular weight is substantially constant in 72 hours.The present invention provides good basis for setting up desirable and reliable AD model, can ensure accuracy and the reliability of experimental result, for the pathogenesis of discussion AD, pathologic process and searching and screening active drug provide effective foundation.
2. the preparation method of A β 1-42 oligomer of the present invention is simple, yield is high, save time, cost is low; Select HEPES buffered soln, the long period can control constant pH scope, obtain better buffering effect compared with F12 damping fluid.
3. adopt SDS-PAGE associated proteins Diagnosis of Sghistosomiasis notation to identify A β 1-42 oligomer of the present invention, the method can not only detect the size of A β 42 monomer or oligomer molecular weight, the purity of A β 42 monomer or oligomer can also be checked simultaneously, with enzyme-linked immunosorbent assay, streaming fluorescence technique, transmission electron microscope observation is compared, there is sensitivity high, high specificity, fast and easy, simple to operate, expense is lower, clear picture, the advantages such as result is accurate, overcome the protein denaturation that current Western blot exists, background is too high, " hangover " phenomenon is there is in electrophoresis process, the problems such as positive band is very weak.
4. the whole testing process of the present invention is all at room temperature carried out, and reduces the steps such as heating, cooling, simplifies whole operating process; Primary antibodie and two anti-be all derive from that its matching of mouse is fabulous, concentration suitable, cross reaction can not be had with encapsulant, two anti-can not to occur in non-specific binding, primary antibodie, containing 6E10, there will not be the phenomenons such as background is high, positive band is very weak in testing process.
Accompanying drawing explanation
Fig. 1 is the protein immunoblot figure of the A β 1-42 oligomer that embodiment 1,2 obtains.
Fig. 2 is Changing Pattern figure in time after the A β 1-42 oligomer synthesis that obtains of embodiment 1.
Fig. 3 is the A β 1-42 oligomer modeling perioperatively stability test figure that embodiment 1 obtains.
Embodiment
Below with reference to specific embodiment, the present invention is further described, but do not limit the scope of the invention and range of application.
One, instrument and reagent
1. key instrument.
(1) Gan Yu Nitrogen evaporator Tianjin Hengao Technology Development Co., Ltd. provides
(2) eppendorf company of shaker Germany provides
(3) invitrogen company of the electrophoresis chamber U.S. provides
(4) invitrogen company of the transferring film instrument U.S. provides
(5) pvdf membrane U.S. Life technologies company provides, article No.: IB401001
2. main agents.
(1) A β 1-42 synthetic peptide, English name: β-Amyloid (1-42), molecular formula C
203h
311n
55o
60s
1molecular weight 4514.1, Chinese Peptide company provides, article No.: AMYD-003, purity more than 96.9%, sequence is as follows: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala.
(2) HFIP, Chinese name: HFIP, German sigma company provides, article No.: 105228.
(3) DMSO, Chinese name: dimethyl sulfoxide (DMSO), German sigma company provides, article No.: D8418.
(4) HEPES, Chinese name: 4-hydroxyethyl piperazine ethanesulfonic acid, German sigma company provides, article No.: H4034-25G.
(5) SDS, Chinese name: sodium lauryl sulphate.
(6) gradient glue of SDS-NuPAGE Novex 4 – 12%Bis-Tris gel: Life technologies company of the U.S. provides, article No.: NP0335BOX.
(7) molecular weight is the pre-dyed molecular weight standards of 3.6-260KD: invitrogen company of the U.S. provides, and commodity are called:
sharp Pre-stained Protein Standard, article No. LC5800.
(8) Tween-20:scientific research special company provides, article No.: 0777.
(9) Tris:Solarbio company provides, article No.: T8060.
(10) skim-milk: scientific research special provides, article No.: 232100.
(11) primary antibodie (amyloid antibody): Convance company of the U.S. provides, commodity are called: β-Amyloid, 1-16 (6E10) mAb, article No.: SIG-39300.
(12) two anti-(sheep anti-mouse iggs of horseradish peroxidase-labeled), Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge provides, trade(brand)name: a Mo IgG (H+L)/HRP, article No.: ZB-2305.
(13) HRP substrate solution: millipore company of the U.S. provides, trade(brand)name: Immobilon tmwestern/chemiluminescon HRP substrate, article No.: WBKLS0100.
3. the preparation of solution.
(1) pH value is the concentration of 7.4 is l0mM HEPES buffered soln: the HEPES getting 0.0238g is dissolved in the distilled water of 10ml, dissolves rear NaOH solution, adjusts the pH to 7.4 of solution.
(2) 4X electrophoresis sample-loading buffer: the 0.5M Tris-HCl (pH6.8) getting 25ml, adds the sucrose of SDS, 10g of 4g, the tetrabromophenol sulfonphthalein of 4mg, then adds distilled water constant volume to 50ml, mixing, to obtain final product.
(3) TBST solution:
0.1M TBS solution: get Tris 60g, NaCl 88g, adds ultrapure water 700ml, and mixing makes dissolving, and with hydrochloric acid adjust pH to 7.5, add water 1000ml, to obtain final product.
TBST solution: get the above-mentioned 0.1M TBS solution 100ml prepared, add 1ml Tween 20, then add ultrapure water to 1000ml, mixing, to obtain final product.
(4) the primary antibodie solution containing 6E10: get primary antibodie 6E10, is diluted to the concentration of 1:5000 with 1% skim milk powder solution.
(5) the two anti-solution containing horseradish peroxidase-labeled: get horseradish peroxidase-labeled two resist, and become the concentration of 1:5000 with the above-mentioned TBST solution dilution prepared.
Two, the preparation of A β 1-42 oligomer
Embodiment 1
A preparation method for A β 1-42 oligomer, comprises the following steps:
1. by the A β 1-42 synthetic peptide of 1mg, add in 220 μ L HFIP, at room temperature leave standstill 30 minutes, obtain consoluet solution;
2. the solution that step 1 obtains is transferred to the centrifuge tube of silication, dry bath Nitrogen evaporator blowing up 8 minutes, HFIP is thoroughly dried up, obtains transparent sheet-like solid and be sunken at the bottom of pipe;
3. in described centrifuge tube, add 40 μ l DMSO, the solid bottom centrifuge tube dissolved completely, then add pH value be 7.4 concentration be l0mM HEPES buffered soln, make the last final volume of solution be 1ml, mix, to obtain final product.
Embodiment 2
A preparation method for A β 1-42 oligomer, comprises the following steps:
1. by the A β 1-42 synthetic peptide of 1mg, add in 225 μ L HFIP, at room temperature leave standstill 60 minutes, obtain consoluet solution;
2. the solution that step 1 obtains is transferred to the centrifuge tube of silication, dry bath Nitrogen evaporator blowing up 15 minutes, HFIP is thoroughly dried up, obtains transparent sheet-like solid and be sunken at the bottom of pipe;
3. in described centrifuge tube, add 40 μ l DMSO, the solid bottom centrifuge tube dissolved completely, then add pH value be 7.4 concentration be l0mM HEPES buffered soln, make the last final volume of solution be 1ml, mix, to obtain final product.
Three, the authentication method of A β 1-42 oligomer
Embodiment 3
An authentication method for A β 1-42 oligomer, comprises the following steps:
1.SDS-polyacrylamide gel electrophoresis: the A β 1-42 oligomer solution 2 μ L that Example 1 obtains, mix with 18 μ L4X electrophoresis sample-loading buffers, then loading, electrophoresis applied sample amount is 5ul/ swimming lane, the glue of electrophoresis is the gradient glue of 4 – 12%, add at a swimming lane pre-dyed molecular weight standards that 10 μ L molecular weight are 3.6-260KD simultaneously, during electrophoresis, select 80V electrophoresis 30 minutes, then turn 100v voltage and continue electrophoresis 2 hours;
2. transferring film: the protein band that electrophoresis is good is transferred on pvdf membrane, semidrying transferring film, the time is 6 minutes, by the size cutting by application of sample swimming lane of the pvdf membrane that takes a turn for the better, to be then dipped in TBST solution 5 minutes by having cut out pvdf membrane;
3. close: be immersed in by pvdf membrane in the TBST solution containing 5% skim-milk, shaking table vibrates, shaking table vibrates at a slow speed, rotating speed is 65 revs/min again, under room temperature condition, close 1 hour;
4. wash film: pvdf membrane step 3 obtained is immersed in TBST solution, shaking table shakes rinsing 2 times, each 5 minutes;
5. primary antibodie is hatched: pvdf membrane step 4 obtained is immersed in the primary antibodie solution containing 6E10, incubated at room temperature 1 hour;
6. wash primary antibodie film: be immersed in by the pvdf membrane of hatching through primary antibodie in TBST solution, shaking table shakes rinsing 6 times, each 5 minutes;
7. two anti-to hatch: the pvdf membrane that rinsing is good is immersed in the two anti-solution containing horseradish peroxidase-labeled, incubated at room temperature 1 hour;
8. wash two anti-films: by being immersed in TBST solution through two anti-pvdf membranes of hatching, shaking table shakes rinsing 6 times, each 5 minutes;
9. chemoluminescence exposure: be evenly added in by HRP substrate solution on the pvdf membrane that step 8 is rinsed, carries out darkroom exposure imaging respectively with the interval of 3 seconds within 5-30 second, therefrom select band clearly film as end-result.
Embodiment 4
An authentication method for A β 1-42 oligomer, comprises the following steps:
1.SDS-polyacrylamide gel electrophoresis: each 2 μ L of A β 1-42 oligomer solution that obtain of Example 1, embodiment 2 respectively, mix with 18 μ L 4X electrophoresis sample-loading buffers, then loading, electrophoresis applied sample amount is 5ul/ swimming lane, the glue of electrophoresis is the gradient glue of 4 – 12%, add at a swimming lane pre-dyed molecular weight standards that 10 μ L molecular weight are 3.6-260KD simultaneously, during electrophoresis, select 80V electrophoresis 40 minutes, then turn 100v voltage and continue electrophoresis 2 hours;
2. transferring film: the protein band that electrophoresis is good is transferred on pvdf membrane, semidrying transferring film, the time is 6 minutes, by the size cutting by application of sample swimming lane of the pvdf membrane that takes a turn for the better, to be then dipped in TBST solution 10 minutes by having cut out pvdf membrane;
3. close: be immersed in by pvdf membrane in the TBST solution containing 5% skim-milk, shaking table vibrates, shaking table vibrates at a slow speed, rotating speed is 75 revs/min again, under room temperature condition, close 1 hour;
4. wash film: pvdf membrane step 3 obtained is immersed in TBST solution, shaking table shakes rinsing 3 times, each 5 minutes;
5. primary antibodie is hatched: pvdf membrane step 4 obtained is immersed in the primary antibodie solution containing 6E10, incubated at room temperature 1 hour;
6. wash primary antibodie film: be immersed in by the pvdf membrane of hatching through primary antibodie in TBST solution, shaking table shakes rinsing 6 times, each 5 minutes;
7. two anti-to hatch: the pvdf membrane that rinsing is good is immersed in the two anti-solution containing horseradish peroxidase-labeled, incubated at room temperature 1 hour;
8. wash two anti-films: by being immersed in TBST solution through two anti-pvdf membranes of hatching, shaking table shakes rinsing 6 times, each 5 minutes;
9. chemoluminescence exposure: be evenly added in by HRP substrate solution on the pvdf membrane that step 8 is rinsed, carries out darkroom exposure imaging respectively with the interval of 5 seconds within 5-30 second, therefrom select band clearly film as end-result.
The qualification result of embodiment 4 is shown in Fig. 1, and in figure, numbering 1,2 is respectively the A β 1-42 oligomer that above-described embodiment 1,2 prepares.As can be seen from the figure: take molecular weight as the obvious band of 12-20KD be master, also there is a small amount of obviously band near 4-4.5KD simultaneously.Show A β 1-42 oligomer of the present invention: main containing 12-20KD oligomer, purity is more than 80%, is tripolymer, the tetramer; Another containing a small amount of 4-4.5KD monomer.
Four, the stability test of A β 1-42 oligomer
Above-mentioned SDS-PAGE associated proteins Diagnosis of Sghistosomiasis notation is adopted to identify the stability of A β 1-42 oligomer of the present invention.
The Time Change test of 1.A β 1-42 oligomer
The A β 1-42 oligomer that above-described embodiment 1 is prepared, preserve under 4 DEG C of conditions, its molecular weight is measured respectively at 0h, 2h, 4h, 6h, 8h, 18h, 24h, 48h, 72h, measurement result is shown in Fig. 2, as can be seen from the figure: in 72h, oligomer molecular weight is substantially constant, illustrate that A β 1-42 oligomer provided by the invention is very stable.
2.A β 1-42 oligomer modeling perioperatively sample stability is tested
The A β 1-42 oligomer prepared by above-described embodiment 1, is expelled in animal model according to weight differences, after terminating, leaves and takes a small amount of sample respectively, for follow-up qualification sample stability, in table 1 before injection with injection.Measurement result is shown in Fig. 3, as can be seen from the figure, prepared obtains A β 1-42 oligomer stable components, is all based on oligomer in surgical procedure, illustrate injection enter solution in animal body mainly oligomer be main, meet the demand of experiment to the injection of A β 1-42 oligomer.
Table 2 A β 1-42 oligomer modeling perioperatively sample stability table
Claims (4)
1. an A β 1-42 oligomer, is characterized in that: described A β 1-42 oligomer is mainly the mixture of oligomer and monomer;
Described oligomer is through Western blot electroresis appraisal: molecular weight is 12-20KD, is tripolymer and the tetramer;
Described monomer is through Western blot electroresis appraisal: molecular weight is 4-4.5KD.
2. A β 1-42 oligomer according to claim 1, it is characterized in that, the preparation method of described A β 1-42 oligomer is: get A β 1-42 synthetic peptide, be at room temperature dissolved completely in HFIP, gained solution is transferred in centrifuge tube, with dry bath Nitrogen evaporator, HFIP is thoroughly dried up; Adding DMSO makes the solid bottom centrifuge tube dissolve completely, adds HEPES damping fluid, mixes, to obtain final product.
3. a preparation method for A β 1-42 oligomer as claimed in claim 1 or 2, is characterized in that, comprise the following steps:
(1) by the A β 1-42 synthetic peptide of 1mg, add in 220-225 μ L HFIP, at room temperature leave standstill 30-60 minute, obtain consoluet solution;
(2) solution that step (1) obtains is transferred to the centrifuge tube of silication, at dry bath Nitrogen evaporator blowing up 8-15 minute, HFIP is thoroughly dried up, obtain transparent sheet-like solid and be sunken at the bottom of pipe;
(3) in described centrifuge tube, add 40 μ l DMSO, the solid bottom centrifuge tube dissolved completely, then add pH value be 7.4 concentration be l0mM HEPES buffered soln, make the last final volume of solution be 1ml, mix, to obtain final product.
4. an authentication method for A β 1-42 oligomer as claimed in claim 1 or 2, is characterized in that, comprise the following steps:
(1) SDS-polyacrylamide gel electrophoresis: get A β 1-42 oligomer solution 2 μ L, mix with 18 μ L 4X electrophoresis sample-loading buffers, then loading, electrophoresis applied sample amount is 5ul/ swimming lane, the glue of electrophoresis is the gradient glue of 4 – 12%, add at a swimming lane pre-dyed molecular weight standards that 10 μ L molecular weight are 3.6-260KD simultaneously, during electrophoresis, select 80V electrophoresis 30-40 minute, then turn 100v voltage and continue electrophoresis 2 hours;
(2) transferring film: the protein band that electrophoresis is good is transferred on pvdf membrane, semidrying transferring film, the time is 6 minutes, by the pvdf membrane that takes a turn for the better by the size cutting of application of sample swimming lane, is then dipped into 5-10 minute in TBST solution by having cut out pvdf membrane;
(3) close: again pvdf membrane is immersed in the TBST solution containing 5% skim-milk, shaking table vibrates, shaking table vibrates at a slow speed, under room temperature condition, close 1 hour;
(4) film is washed: be immersed in TBST solution by the pvdf membrane that step (3) obtains, shaking table shakes rinsing 2-3 time, each 5 minutes;
(5) primary antibodie is hatched: be immersed in by the pvdf membrane that step (4) obtains in the primary antibodie solution containing 6E10, incubated at room temperature 1 hour;
(6) wash primary antibodie film: be immersed in by the pvdf membrane of hatching through primary antibodie in TBST solution, shaking table shakes rinsing 6 times, each 5 minutes;
(7) two anti-hatch: be immersed in by the pvdf membrane that rinsing is good in the two anti-solution containing horseradish peroxidase-labeled, incubated at room temperature 1 hour;
(8) two anti-films are washed: by being immersed in TBST solution through two anti-pvdf membranes of hatching, shaking table shakes rinsing 6 times, each 5 minutes;
(9) chemoluminescence exposure: be evenly added in by HRP substrate solution on the pvdf membrane that step (8) is rinsed, carries out darkroom exposure imaging respectively within 5-30 second, therefrom select band clearly film as end-result.
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| EP1944314A1 (en) * | 2007-01-11 | 2008-07-16 | Philipps-Universität Marburg | Diagnosis of Alzheimer's disease and other neurodementing disorders |
| CN101948523A (en) * | 2010-08-26 | 2011-01-19 | 北京交通大学 | Artificial in-vitro preparation method of low molecular weight amyloid peptide oligomer and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1944314A1 (en) * | 2007-01-11 | 2008-07-16 | Philipps-Universität Marburg | Diagnosis of Alzheimer's disease and other neurodementing disorders |
| CN101948523A (en) * | 2010-08-26 | 2011-01-19 | 北京交通大学 | Artificial in-vitro preparation method of low molecular weight amyloid peptide oligomer and application |
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| 张钧田等: "《现代药理试验方法上》", 31 July 2012 * |
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