CN109239169A - A kind of transferring film method of electrophoretic gel mobility experiment - Google Patents
A kind of transferring film method of electrophoretic gel mobility experiment Download PDFInfo
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- CN109239169A CN109239169A CN201810954665.4A CN201810954665A CN109239169A CN 109239169 A CN109239169 A CN 109239169A CN 201810954665 A CN201810954665 A CN 201810954665A CN 109239169 A CN109239169 A CN 109239169A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The invention discloses a kind of transferring film methods of electrophoretic gel mobility experiment to be cut to filter paper and nylon membrane and the gel same size that is, according to gel size；According to filter paper-nylon membrane-gel-filter paper sequence, experimental material is placed from bottom to top, is prepared into transferring film block；The transferring film liquid 1-5ml of the Fresh after 4 DEG C of refrigerations is uniformly sprayed on transferring film block, after transferring film block be put into half-dried transferring film instrument complete transferring film.The present invention provides a kind of transferring film methods of electrophoretic gel mobility experiment, overcome conventional wet and turn, take a long time, and reagent consumption is big, the low technological deficiency of overall efficiency, transferring film can be completed in 15min by having reached, the purpose for improving transferring film efficiency has effect of optimization to EMSA experiment.
Description
Technical field
The present invention relates to protein detection technology fields, more particularly to a kind of transferring film method of electrophoretic gel mobility experiment.
Background technique
Different components is distinguished by electrophoresis, and is transferred to solid support, by specific reagent (antibody) as spy
Needle detects target substance, and the western blot technology of protein combines the high-resolution and solid-phase immunity of gel electrophoresis
Measurement detects target substance, and the western blot technology of protein combines the high-resolution of gel electrophoresis and solid phase is exempted from
Epidemic disease measurement.
It wherein under the action of electric current, is transferred to protein on solid phase carrier (film) from glue, is the Western of protein
The core link of engram technology, still, in traditional electrophoretic gel mobility experiment (EMSA), need by gel protein with
Probe is transferred on nylon membrane, to complete subsequent electrochemiluminescence assay.Usually used electrotransfer method is wet turn, voltage
Between 100v-120v, the transferring film time is 30min-60min, is taken a long time, and reagent consumption is big, overall efficiency is low.
Therefore, a kind of transferring film method for inventing electrophoretic gel mobility experiment, sufficiently improves transferring film efficiency, when reducing transferring film
Between, the problem of there is effect of optimization, be those skilled in the art's urgent need to resolve to the overall flow of experiment.
Summary of the invention
In view of this, transferring film efficiency is sufficiently improved the present invention provides a kind of transferring film method of electrophoretic gel mobility experiment,
The transferring film time is reduced, there is effect of optimization to the overall flow of experiment, can be applied to EMSA experiment and Southern
In the experiment of Blotting.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of transferring film method of electrophoretic gel mobility experiment, comprising the following steps:
(1) according to gel size, filter paper and nylon membrane are cut to and the gel same size；
(2) experimental material is wherein placed from bottom to top according to filter paper-nylon membrane-gel-filter paper sequence, be prepared into and turn
Film block；
(3) by the transferring film liquid 1-5ml of the Fresh after 4 DEG C of refrigerations, uniformly it is sprayed at transferring film block described in step (2)
On, after the transferring film block is put into half-dried transferring film instrument；
(4) instrument switch is opened, setting transferring film voltage is 25-30V, continuous current 1A-0.3A, and the transferring film time is
5min-15min；
(5) electrochemiluminescence assay is carried out after the completion of transferring film and gel imaging system is taken pictures.
The present invention provides a kind of transferring film methods of electrophoretic gel mobility experiment, overcome conventional wet and turn, take a long time,
And reagent consumption is big, the low technological deficiency of overall efficiency, transferring film can be completed in 15min by having reached, and improve transferring film efficiency
Purpose.
Further, step (2) method particularly includes: three layers of filter paper are first layered on bottom, and remove bubble；Again by institute
It states nylon membrane paper to be layered on the filter paper, and removes bubble；By the gel overlay on the nylon membrane, and remove bubble；Finally
Three layers of filter paper are covered on the gel again.
Wherein filter paper, glue, cannot have bubble between film, the formation of bubble will cause short circuit.
Further, the transferring film liquid is 0.5 × TBE.
A kind of transferring film method of electrophoretic gel mobility experiment EMSA test or Southern Blotting experiment in answer
With.
It can be seen via above technical scheme that compared with prior art, it is real that the present invention provides a kind of electrophoretic gel mobilities
The transferring film method tested has following technological merit:
The present invention provides a kind of transferring film methods of electrophoretic gel mobility experiment, overcome conventional wet and turn, take a long time,
And reagent consumption is big, the low technological deficiency of overall efficiency, transferring film can be completed in 15min by having reached, and improve transferring film efficiency
Purpose, to EMSA experiment have effect of optimization.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is the experimental result of conventional wet robin provided by the invention；
Wherein swimming lane 1 is Biotin-EBNA Control DNA；Swimming lane 2 is Biotin-EBNA Control DNA+
EBNA extract；Swimming lane 3 is Biotin-EBNA Control DNA+EBNA extract+200-fold molar
excess of unlabeled EBNA DNA；
Fig. 2 attached drawing is the experimental result using transferring film method provided by the invention；
Wherein swimming lane 1 is Biotin-EBNA Control DNA；Swimming lane 2 is Biotin-EBNA Control DNA+
EBNA extract+200-fold molar excess of unlabeled EBNA DNA；Swimming lane 3 is Biotin-EBNA
Control DNA+EBNA extract；
Fig. 3 attached drawing is to detect transcription factor AP -1 in THP-1 cell using transferring film method provided by the invention；
Wherein band 1 is position of the AP-1 probe in conjunction with nucleoprotein, and 2 position of band is in place for free AP-1 probe institute
It sets；
Fig. 4 attached drawing is to detect transcription factor NF-Kb in THP-1 cell using transferring film method provided by the invention；
Wherein band 1 is position of the NF-Kb probe in conjunction with nucleoprotein, and 2 position of band is in place for free NF-Kb probe institute
It sets.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The embodiment of the invention discloses a kind of transferring film methods of electrophoretic gel mobility experiment will filter that is, according to gel size
Paper and nylon membrane are cut to the shape with gel same size；According to filter paper-nylon membrane-gel-filter paper sequence, from bottom to top
Experimental material is placed, transferring film block is prepared into；Three layers of filter paper are first wherein layered on bottom, and remove bubble；Again by the nylon
Film paper is layered on the filter paper, and removes bubble；By the gel overlay on the nylon membrane, and remove bubble；Finally again by three
The layer filter paper is covered on the gel.0.5 × TBE of transferring film liquid is prepared, 4 DEG C of prepared transferring film liquid are refrigerated, and draws
The transferring film liquid of 3ml is uniformly sprayed on transferring film block, after transferring film block is put into half-dried transferring film instrument.Instrument switch is then turned on, if
Setting transferring film voltage is 25V, and continuous current 0.3A, the transferring film time is 15min, carry out after the completion of transferring film electrochemiluminescence assay and
Gel imaging system is taken pictures.Above-mentioned transferring film method is applied in EMSA is tested or Southern Blotting is tested.
Embodiment 2
The embodiment of the invention discloses a kind of transferring film methods of electrophoretic gel mobility experiment will filter that is, according to gel size
Paper and nylon membrane are cut to the shape with gel same size；According to filter paper-nylon membrane-gel-filter paper sequence, from bottom to top
Experimental material is placed, transferring film block is prepared into；Three layers of filter paper are first wherein layered on bottom, and remove bubble；Again by the nylon
Film paper is layered on the filter paper, and removes bubble；By the gel overlay on the nylon membrane, and remove bubble；Finally again by three
The layer filter paper is covered on the gel.0.5 × TBE of transferring film liquid is prepared, 4 DEG C of prepared transferring film liquid are refrigerated, and draws
The transferring film liquid of 1ml is uniformly sprayed on transferring film block, after transferring film block is put into half-dried transferring film instrument.Instrument switch is then turned on, if
Setting transferring film voltage is 30V, and continuous current 0.8A, the transferring film time is 5min, carry out after the completion of transferring film electrochemiluminescence assay and
Gel imaging system is taken pictures.Above-mentioned transferring film method is applied in EMSA is tested or Southern Blotting is tested.
Embodiment 3
The embodiment of the invention discloses a kind of transferring film methods of electrophoretic gel mobility experiment will filter that is, according to gel size
Paper and nylon membrane are cut to the shape with gel same size；According to filter paper-nylon membrane-gel-filter paper sequence, from bottom to top
Experimental material is placed, transferring film block is prepared into；Three layers of filter paper are first wherein layered on bottom, and remove bubble；Again by the nylon
Film paper is layered on the filter paper, and removes bubble；By the gel overlay on the nylon membrane, and remove bubble；Finally again by three
The layer filter paper is covered on the gel.0.5 × TBE of transferring film liquid is prepared, 4 DEG C of prepared transferring film liquid are refrigerated, and draws
The transferring film liquid of 5ml is uniformly sprayed on transferring film block, after transferring film block is put into half-dried transferring film instrument.Instrument switch is then turned on, if
Transferring film voltage is set as 27V, continuous current 1A, the transferring film time is 8min, and electrochemiluminescence assay is carried out after the completion of transferring film and is coagulated
Glue imaging system is taken pictures.Above-mentioned transferring film method is applied in EMSA is tested or Southern Blotting is tested.
Embodiment 4
The present embodiment is with ThermoQuality-control product in Chemiluminescent EMSA Kit, point
Not using in embodiment 1 transferring film method and traditional wet process transferring film method tested, as a result such as Fig. 1-2.Wherein wet process transferring film
The specific steps of method are as follows:
By Fig. 1-2 it is found that two kinds of transferring film method transferring film results are consistent, further illustrating half-dried transferring film method can be applied
It is tested in EMSA, it might even be possible in the experiment applied to Southern Blotting.This method more saves the time, improves
Conventional efficient.
Embodiment 5
The present embodiment is with transcription factor AP -1 and transcription factor NF- in the transferring film method detection THP-1 cell in embodiment 3
Kb, as a result as shown in Figure 3-4.
The present invention provides a kind of transferring film methods of electrophoretic gel mobility experiment, overcome conventional wet and turn, take a long time,
And reagent consumption is big, the low technological deficiency of overall efficiency, transferring film can be completed in 15min by having reached, and improve transferring film efficiency
Purpose, to EMSA experiment have effect of optimization.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so being described relatively simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (4)
1. a kind of transferring film method of electrophoretic gel mobility experiment, which comprises the following steps:
(1) according to gel size, filter paper and nylon membrane are cut to and the gel same size；
(2) wherein experimental material is placed from bottom to top, is prepared into transferring film block according to filter paper-nylon membrane-gel-filter paper sequence；
(3) it by the transferring film liquid 1-5ml of the Fresh after 4 DEG C of refrigerations, is uniformly sprayed on transferring film block described in step (2), after
The transferring film block is put into half-dried transferring film instrument；
(4) instrument switch is opened, setting transferring film voltage is 25-30V, and continuous current 1A-0.3A, the transferring film time is 5min-
15min；
(5) electrochemiluminescence assay is carried out after the completion of transferring film and gel imaging system is taken pictures.
2. a kind of transferring film method of electrophoretic gel mobility experiment according to claim 1, which is characterized in that step (2)
Method particularly includes: three layers of filter paper are first layered on bottom, and remove bubble；The nylon membrane paper is layered on the filter paper again,
And remove bubble；By the gel overlay on the nylon membrane, and remove bubble；Finally three layers of filter paper are covered on again described
On gel.
3. a kind of transferring film method of electrophoretic gel mobility experiment according to claim 1, which is characterized in that the transferring film liquid
For 0.5 × TBE.
4. a kind of transferring film method of electrophoretic gel mobility experiment according to claim 1 is in EMSA experiment or Southern
It is applied in Blotting experiment.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810954665.4A CN109239169A (en) | 2018-08-21 | 2018-08-21 | A kind of transferring film method of electrophoretic gel mobility experiment |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810954665.4A CN109239169A (en) | 2018-08-21 | 2018-08-21 | A kind of transferring film method of electrophoretic gel mobility experiment |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101241140A (en) * | 2008-03-13 | 2008-08-13 | 上海交通大学 | Quantum dot mark based immune blotting detection method |
| CN106771141A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of rapid assay methods of brucellosis |
| CN107406481A (en) * | 2015-03-03 | 2017-11-28 | 生物辐射实验室股份有限公司 | Electrophoresis and electroblotting system and method |
| US20170356876A1 (en) * | 2015-11-10 | 2017-12-14 | Woodham Biotechnology Holdings, LLC | Gel Electrophoresis and Transfer Combination using Conductive Polymers and Method of Use |
-
2018
- 2018-08-21 CN CN201810954665.4A patent/CN109239169A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101241140A (en) * | 2008-03-13 | 2008-08-13 | 上海交通大学 | Quantum dot mark based immune blotting detection method |
| CN107406481A (en) * | 2015-03-03 | 2017-11-28 | 生物辐射实验室股份有限公司 | Electrophoresis and electroblotting system and method |
| US20170356876A1 (en) * | 2015-11-10 | 2017-12-14 | Woodham Biotechnology Holdings, LLC | Gel Electrophoresis and Transfer Combination using Conductive Polymers and Method of Use |
| CN106771141A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of rapid assay methods of brucellosis |
Non-Patent Citations (2)
| Title |
|---|
| MATHIEU GOUMAIN 等: "Y Receptors Characterized by RT-PCR", 《METHODS IN MOLECULAR BIOLOGY， VOLUME 153： NEUROPEPTIDE Y PROTOCOLS》 * |
| 张申 等主编: "《分子生物学检验》", 31 January 2017 * |
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