CN102095867A - Application of self-luminescent material to chemiluminescence western blot and composition of self-luminescent material - Google Patents
Application of self-luminescent material to chemiluminescence western blot and composition of self-luminescent material Download PDFInfo
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- CN102095867A CN102095867A CN2009102001811A CN200910200181A CN102095867A CN 102095867 A CN102095867 A CN 102095867A CN 2009102001811 A CN2009102001811 A CN 2009102001811A CN 200910200181 A CN200910200181 A CN 200910200181A CN 102095867 A CN102095867 A CN 102095867A
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Abstract
The invention discloses novel application of a self-luminescent material to the display of a protein molecular weight marker on an X-ray film in a chemiluminescence western blot experiment. The invention also discloses a self-luminescent material-containing composition which comprises the self-luminescent material, a coupling agent and a filler, and an improved chemiluminescence western blot method for displaying the protein molecular weight marker on the X-ray film. In the conventional chemiluminescence western blot operation process, the reagent is coated at the position of the pre-dyed protein molecular weight marker before an X-ray plate is exposed, and a clear protein molecular weight marker developing strip can be obtained on the X-ray plate after the X-ray plate is exposed, so that the aim of measuring a target protein strip is fulfilled, the defects of the conventional chemiluminescence western blot are overcome, and the western blot technology is greatly improved. The self-luminescent material is widely applied to scientific research experiments; and the preparation method of the used reagent is environmental-friendly and does not have any toxic effect on human bodies.
Description
Technical field
The invention belongs to biological technical field, particularly self-luminescent material is used for showing protein molecular weight standard and composition and Westernblot method on the x-ray film of chemiluminescence Western blot.
Background technology
At present, whether a certain protein expression level changes in research aircraft soma or the cell, nearly all needs to use chemiluminescence protein immunoblotting (Western blot) method to detect.But this method testing result only can show detected band on the X-film after the development, can not show the protein molecular weight standard of an accurate quantification destination protein simultaneously, weighs the molecular weight size of detected albumen.Though this is that it can the intensity uniform stripe occur when electrophoresis, understand the position of destination protein easily because added pre-dsred protein molecular weight standard in the track when SDS-PAGE; Can also monitor the transfer efficiency of follow-up immunization trace.But, also be short of most exactly can not the luminous Western blot of quantitative chemical X-ray film on the molecular weight of the band that occurs, if molecular weight changed after glycosylation or phosphorylation modification had taken place especially detected albumen.This shortcoming is that the luminous Western blot of conventional chemical method is indeterminable, in addition do not have yet so far science easy, add the method for protein molecular weight standard intuitively.Therefore need a kind of method that is used for interpolation protein molecular weight standard on the chemiluminescence Western blot experiment X-ray film badly.
Usually Western blot method step is: by the protein of SDS-PAGE separation, transfer on NC or the pvdf membrane, treat that with the energy specific recognition antibody of Reichl's test reacts then, after washing is removed and is not had the specific antibody of combination, it is anti-to add species specificity two marks such as horseradish peroxidase or alkaline phosphatase, energy identification specificity antibody, wash the labelled antibody of removing non-specific binding after reaction a period of time once more, can be luminous after reactions such as colour developing liquid and horseradish peroxidase or alkaline phosphatase.Be pressed on NC or the pvdf membrane with the X-film this moment and expose, will stay the image of destination protein on the film after the flushing.If the destination protein amount is big, the protein antibodies compound of its formation is just big, and light is just bright, the image on the film just big and and color depth; Otherwise it is little with regard to band, look shallow.Can judge with this whether the destination protein expression changes.But shortcoming of this classic method is exactly, the pre-dsred protein molecular weight standard that uses when doing Western blot experiment can only monitor the destination protein electrophoretic migration before the albumen transfer printing, transfer efficiency, this is because pre-dsred protein molecular weight standard can not develop on the X-film, it goes up the molecular weight size of the protein band of appearance so can not estimate X-film colour developing back, an if more than strip-like developing pipe on the X-film of colour developing back, that just can not illustrate accurately which bar is that the destination protein band develops, which is a series of contradictions such as non-specific band, more quantitatively destination protein generation phosphorylation, molecular weight changes and occurs the problem of experimental phenomenas such as a plurality of bands after the glycosylation.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly at the deficiency that can not show protein molecular weight standard on the x-ray film in the existing chemiluminescence Western blot technology, and a kind of method and compositions for use thereof that makes the x-ray film demonstration protein molecular weight standard in the experiment of chemiluminescence protein immunoblotting is provided.
For solving the problems of the technologies described above, the invention provides the application in the x-ray film demonstration protein molecular weight standard of self-luminescent material in the experiment of chemiluminescence protein immunoblotting.Reagent especially for protein molecular weight standard on the x-ray film in the experiment of preparation demonstration chemiluminescence protein immunoblotting.
The present invention also provides a kind of x-ray film that is used for making the experiment of chemiluminescence protein immunoblotting to show the composition of protein molecular weight standard, comprises self-luminescent material, coupling agent and filling agent.
Among the present invention, described self-luminescent material comprises emissive type luminescent material and light storage type self luminous material.The basis of emissive type luminescent material is radioactive material, does not need to absorb energy from the outside, and night or daytime are all sustainable luminous, are called first generation self-luminescent material again.Light storage type self luminous material is initiatively inhaled visible lights such as holding sunshine, light, ultraviolet light, parasitic light, just can continue luminous in the dark.Be mainly traditional all-sulphide phosphor and development in recent years and with the rare earth compound be faster matrix and be the luminescent material of activator with the rare earth element.Traditional all-sulphide phosphor is a second generation self-luminescent material, and luminosity is low, and the duration is short.Be matrix with the rare earth compound and be that the luminescent material of activator is a third generation self-luminescent material with the rare earth element.This material just can continue more than the luminous 12h after initiatively inhaling visible light 5～10min such as holding sunshine, light, ultraviolet light, parasitic light in the dark, and can make it send multiple coloramas such as red, green, blue, Huang, purple according to actual needs.The light storage type self luminous material of third generation rare earth, chemical property is stable, extinction, hold light, luminescence process can repeat, can reach more than 20 years serviceable life, and nonhazardous, do not contain radioactivity, production run also unharmful substance produces, for the present invention preferred.The preferred 4SrO.7AL of the present invention
2O
3: Eu, 4SrO.7AL
2O
3: Dy, 4SrO.7AL
2O
3: La, CaAl
2O
4: Eu, CaAl
2O
4: Nb, Sr
4Al
14O
25: Eu, Sr
4Al
14O
25: Dy, SrAl
2O
4: Eu, SrAl
2O
4: Dy or their potpourri.What the content of self-luminescent material in composition was preferable is 5%～35%, more preferably 15%～25%, and number percent is mass percent.
Among the present invention, described coupling agent is the important composition composition, plays the dispersion self-luminescent material, gives composition with suitable viscosity, flowability and absorption property, can be bonded to the surface of NC film or pvdf membrane after use.Described coupling agent is glycerine or resin preferably.Described resin comprises various natural resins and synthetic resin.What the content of coupling agent in composition was preferable is 50%～85%, more preferably 65%～70%, and number percent is mass percent.
Among the present invention, described filling agent is white, transparent, translucent, as not hide self-luminescent material absorption or the material that discharges light, mainly plays packing action.Suitably adopt filling agent, both can reduce the self-luminescent material consumption, reduce cost, the character of adjustable compositions again is as rare thick, flowability etc.Filling agent preferably clear lacquer, the acrylic acid clear lacquer, the natural resin clear lacquer or their potpourri among the present invention.What the content of filling agent in composition was preferable is 5%～30%, more preferably 10%～15%, and number percent is mass percent.
Composition of the present invention, the preferable auxiliary element that can further include is as drying agent, hardening agent etc.
One preferred embodiment of composition of the present invention is that the component of quality percentage composition is as follows: 20%4SrO.7AL
2O
3: Eu, 70% glycerine, 2% resin and 8% clear lacquer.
Composition of the present invention drying at room temperature is for a long time preserved.
Among the present invention, described protein immunoblotting claims Western blot again, is this area method commonly used.
The x-ray film that is used to make the experiment of chemiluminescence protein immunoblotting of the present invention shows that the preparation of compositions method of protein molecular weight standard is each component simply to be mixed to get final product as the reagent of routine.
The composition that the x-ray film that is used for making the experiment of chemiluminescence protein immunoblotting of the present invention shows protein molecular weight standard store, transportation and during as commodity selling, can be placed on one preserves in the pipe, this preserves the pipe body is conical, the bottom is taper, there is lid in pipe portion, and scale is arranged on the tube wall.Described lid is screw top preferably.
The present invention also provides a kind of method that makes the x-ray film demonstration protein molecular weight standard in the experiment of chemiluminescence protein immunoblotting, comprise and to carry out video picture from NC film or the pvdf membrane that running gel has shifted the protein albumen that comprises pre-dsred protein molecular weight standard at x-ray film, it is characterized in that, the aforesaid composition of coating carries out the x-ray film video picture then on the band position that shows pre-dsred protein molecular weight standard on described NC film or the pvdf membrane.
Among the present invention, described NC film (cellulose acetate membrane) or pvdf membrane (nitrocellulose filter) are that protein immunoblotting protein commonly used shifts film.It has shifted protein and pre-dsred protein molecular weight standard, has carried out chemiluminescence reaction, need carry out next step X-ray video picture.Described pre-dsred protein molecular weight standard is the protein and the dyestuff covalency coupling matter of various known different molecular weights, can directly be observed visually in electrophoresis process or when changeing film.
Can get the aforesaid composition of the present invention by utilizing pen or pen core shape object to dip in, said composition is coated on NC film or the pvdf membrane.Composition of the present invention can be coated on each band of pre-dsred protein molecular weight standard accurately, and can diffuse pollution not arrive non-target location.
Described NC film or pvdf membrane carry out the method for video picture and can carry out according to the method among the chemiluminescence Western blot of routine on x-ray film.Because the composition that is coated with on NC film or the pvdf membrane has the characteristic of nature Weak-luminescence, can develop when therefore on the film of X-ray, exposing, thereby accurately pre-dsred protein molecular weight standard mark is presented on the film, can be used for the molecular weight size of video picture band on the quantitative x-ray film then.
The present invention also provides a kind of method of chemiluminescence protein immunoblotting of improvement, comprise and to carry out video picture from NC film or the pvdf membrane that running gel has shifted the protein albumen that comprises pre-dsred protein molecular weight standard at x-ray film, it is characterized in that, the aforesaid composition of coating carries out the x-ray film video picture then on the band position that shows pre-dsred protein molecular weight standard on described NC film or the pvdf membrane.Other conditions of described chemiluminescence protein immunoblotting as mentioned above.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
Than prior art, beneficial effect summation of the present invention is as follows: the present invention is by in the luminous Western blot of conventional chemical operating process, spread upon pre-dsred protein molecular weight standard position with reagent of the present invention before the exposure of X-ray mating plate, the exposure back just can obtain protein molecular weight standard strip-like developing pipe clearly on the X-ray mating plate, reach the purpose of measuring the destination protein band, effectively remedy the deficiency of the luminous Western blot of conventional chemical, improved Western blot technology greatly.The protein molecular weight standard band that adds is clear, meets magazine molecular weight mark standard in the world, is widely used in scientific experiment.The present invention also provides preparation method of reagent thereof that this method is used, and environmental protection, human body is not had any toxic action.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 shows the reagent of protein molecular weight standard for the present invention one preferably is used for making the x-ray film of chemiluminescence protein immunoblotting experiment.
Fig. 2 is the gel figure behind the SDS-PAGE electrophoresis among the Western blot, and use therein is pre-dsred protein molecular weight standard.
Fig. 3 is the NC film picture that carries pre-dsred protein molecular weight standard among the Western blot behind the SDS-PAGE commentaries on classics film.
Fig. 4 among the Western blot after reagent of the present invention is smeared in the band position of pre-dsred protein molecular weight standard on the NC of the transfer printing destination protein film picture.
Fig. 5 among the Western blot on the NC film band position of pre-dsred protein molecular weight standard smeared the picture of reagent exposure of the present invention back x-ray film.Wherein do not use reagent of the present invention to be reference examples.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the present invention is meant the temperature of the operation room of testing, and is generally 25 ℃.Described number percent part unless otherwise noted is meant mass percent.
Prescription:
| Raw material | Production firm | Addition/mg |
| 4SrO·7AL 2O 3:Eu | Guangzhou rare metal research institute | 20 |
| Glycerine | Shanghai Inst of Chemical Reagent | 70 |
| Resin | The bright Chemical Factory of Daily Use in Zibo City | 2 |
| The clear lacquer | Qingdao dust bird with red feathers lacquer industry company limited | 8 |
Also high-speed stirred is extremely even to get each raw material mixing by prescription, promptly gets the oily transparency liquid, and nothing is smelt, little apparent yellow green.
See Fig. 1.Preserve pipe 1 for one, its body is a taper shape 2, and the bottom is taper 3, and there is screw top 4 in pipe portion, and scale 5 is arranged on the tube wall, preserves in the pipe above-mentioned composition is housed.
β in the U2OS cell-actin albumen Western blot detects
(1) extract the total protein that is incubated at the U2OS cell (available from cell resource center of Shanghai Sheng Ke institute of the Chinese Academy of Sciences) on the 6 porocyte culture plates, BCA kit (giving birth to the worker available from Shanghai) is measured and is extracted total protein of cell concentration.
(2) SDS-PAGE electrophoresis: can SDS-PAGE running gel, resolving gel concentration adopts 12%, concentrates gum concentration and adopts 5%.The cell protein and the 2 * SDS damping fluid (100mmol/LTris-HCl (pH6.8), 200mmol/L dithiothreitol (DTT) (DTT), 4%SDS, 0.2% bromophenol blue, 20% glycerine) that extract are mixed with volume, boil 5min.The albumen applied sample amount is 20 μ g/ swimming lanes.After sample adds well, in well subsequently, add 3 μ L and dye molecular weight of albumen standard (Canadian Fermentas company produce) in advance.Electrophoretic voltage is constant voltage 90V in the concentrated glue, and the separation gel electrophoretic voltage is constant voltage 120V, finishes electrophoresis when bromophenol blue is gone out separation gel fully, and the gel picture is seen Fig. 2 behind the electrophoresis.
(3) electricity changes: with the electrophoretic blotting groove albumen on the gel being wet goes on the NC film, and the NC film picture behind the commentaries on classics film is seen Fig. 3, and pre-dsred protein molecular weight standard is high-visible on the NC film.
(4) sealing and antibodies: the NC film seals 2h with 5% (wt) skimmed milk room temperature, then with the NC film respectively with anti-human actin (the monoclonal antibody incubated at room 2h of β-actin).With TBST (25mM Tris, 140mM sodium chloride, 3mM potassium chloride, 0.05% Tween-20) washing 3 times, each 10min is with rabbit anti-mouse igg (1: 5000 times of dilution) the room temperature reaction 2h of peroxidase labelling.TBST washing 3 times, each 10min.
(5) before the colour developing by following processing: utilize pen or simple pen core shape object to dip in the reagent of getting embodiment 1 preparation, evenly spread upon on the band of pre-dsred protein molecular weight standard of NC film, the NC film is seen Fig. 4 after handling.As seen, the reagent of the present invention preparation can be firm attached on the NC film that has soaked into the experiment aqueous solutions employed, and keep stable, just as simply and stablely do not fade with on paper, writing, not dizzy dying.The contrast NC film of not using above-mentioned agent treated is set simultaneously.
(6) colour developing: chemical illuminating reagent colour developing, use Super Signal West PicoChemiluminescent Substrate Kit (chemical luminescence reagent kit is available from U.S. Thermo Scientific company), according to its instructions, with the exposure of X-ray film darkroom, scanner scanning film.Film scanning figure sees Fig. 5.Wherein, no protein molecular weight standard shows in the blank, shows protein molecular weight standard and smeared on the film of self-luminescent material composition of the present invention.As seen, use the inventive method adding accurate protein molecular weight standard in the chemiluminescence Western blot experimental result arbitrarily.This has remedied the quantitatively Western blot problem of band molecular weight size as a result of classic method.Owing to there is protein molecular weight standard in the result, modifying (glycosylation, phosphorylation and ubiquitinization etc.) for destination protein among the qualitative Western blot result has important indicative significance.
According to the form below 1 prescription is prepared the composition of various self-luminescent materials by the method for embodiment 1, is used for Western blot experiment and shows protein molecular weight standard on the X-film, and method of operating is with embodiment 2.As a result each composition all can on NC film or pvdf membrane, smear evenly, stick firmly, not fade, not dizzy dying.The protein molecular weight standard border that shows on the X-film is clearly demarcated, clear, regular.
Table 1. composite formula
Claims (10)
1. the x-ray film of self-luminescent material in the experiment of chemiluminescence protein immunoblotting shows the application in the protein molecular weight standard.
2. application as claimed in claim 1 is characterized in that, self-luminescent material is used for preparing the reagent that shows protein molecular weight standard on the chemiluminescence protein immunoblotting experiment x-ray film.
3. application as claimed in claim 1 is characterized in that, described self-luminescent material is a third generation self-luminescent material.
4. an x-ray film that is used for making the experiment of chemiluminescence protein immunoblotting shows the composition of protein molecular weight standard, it is characterized in that, comprises self-luminescent material, coupling agent and filling agent.
5. composition as claimed in claim 4 is characterized in that, described self-luminescent material is a third generation self-luminescent material; Described coupling agent is glycerine or resin; Described filling agent is the clear lacquer, the acrylic acid clear lacquer, the natural resin clear lacquer or their potpourri.
6. composition as claimed in claim 5 is characterized in that, described third generation self-luminescent material is 4SrO.7AL
2O
3: Eu, 4SrO.7AL
2O
3: Dy, 4SrO.7AL
2O
3: La, CaAl
2O
4: Eu, CaAl
2O
4: Nb, Sr
4Al
14O
25: Eu, Sr
4Al
14O
25: Dy, SrAl
2O
4: Eu, SrAl
2O
4: Dy or their potpourri.
7. composition as claimed in claim 4 is characterized in that, the content of described self-luminescent material is 5%～35%, and the content of described coupling agent is 50%～85%, and the content of described filling agent is 5%～30%, and number percent is mass percent.
8. composition as claimed in claim 4 is characterized in that the component of described composition is as follows: 20%4SrO.7AL
2O
3: Eu, 70% glycerine, 2% resin and 8% clear lacquer, number percent are mass percent.
9. method that makes x-ray film in the chemiluminescence protein immunoblotting experiment show protein molecular weight standard, comprise and to carry out video picture from NC film or the pvdf membrane that running gel has shifted the protein albumen that comprises pre-dsred protein molecular weight standard at x-ray film, it is characterized in that, coating is carried out the x-ray film video picture then as each described composition of claim 4～8 on the band position that shows pre-dsred protein molecular weight standard on described NC film or the pvdf membrane.
10. the method for a chemiluminescence protein immunoblotting, comprise and to carry out video picture from NC film or the pvdf membrane that running gel has shifted the protein albumen that comprises pre-dsred protein molecular weight standard at x-ray film, it is characterized in that, coating is carried out the x-ray film video picture then as each described composition of claim 4～8 on the band position that shows pre-dsred protein molecular weight standard on described NC film or the pvdf membrane.
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| CN102095867B CN102095867B (en) | 2013-11-27 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20180027538A (en) * | 2015-07-08 | 2018-03-14 | 상하이 이-블롯 포토일렉트릭 테크놀로지 컴퍼니 리미티드 | Method and apparatus for signal acquisition of photosensitive chip and method and apparatus for cell tracking |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI518316B (en) | 2014-04-01 | 2016-01-21 | 財團法人工業技術研究院 | Optical readout imaging system and biochemical detection method using the same |
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| KR102084233B1 (en) | 2015-07-08 | 2020-03-03 | 상하이 이-블롯 포토일렉트릭 테크놀로지 컴퍼니 리미티드 | Signal acquisition method and device of photosensitive chip and cell tracking method and device |
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| CN102095867B (en) | 2013-11-27 |
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