CN102636548A - Method for detecting genetic polymorphism of transferrin through gradient gel electrophoresis - Google Patents
Method for detecting genetic polymorphism of transferrin through gradient gel electrophoresis Download PDFInfo
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- CN102636548A CN102636548A CN2012101399838A CN201210139983A CN102636548A CN 102636548 A CN102636548 A CN 102636548A CN 2012101399838 A CN2012101399838 A CN 2012101399838A CN 201210139983 A CN201210139983 A CN 201210139983A CN 102636548 A CN102636548 A CN 102636548A
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Abstract
The invention discloses a method for detecting genetic polymorphism of transferrin through gradient gel electrophoresis. According to the method, the polymorphism of the transferrin is detected by using horizontal plate polyacrylamide gradient gel electrophoresis, wherein the gel concentrations of horizontal plate polyacrylamide gradient gel from the top to the bottom are 12 percent, 8 percent and 6 percent respectively; and the electrophoresis conditions are that pre-electrophoresis is performed by using voltage of 380 V for 10-15 minutes and then electrophoresis is performed by using voltage of 740 V for 5-6 hours. By applying the method provided by the invention, the transferrin can be separated well, the band is clear, no interference phenomenon is caused, a large number of samples can be simultaneously detected, and the defects of unclear vertical plate gel electrophoresis band, low parting efficiency and the like are overcome. Thus, the horizontal plate polyacrylamide gradient gel electrophoresis adopted in the invention has the characteristics of simplicity, convenience, quickness, sensitivity and the like; and the method is a new detection method provided for detecting the genetic polymorphism of the transferrin of a sheep.
Description
Technical field
With the method for leveling board polyacrylamide gel gradient electrophoresis detection transferrin polymorphism, relate to the genetic polymorphism that the defective of using the gradient electrophoresis technology to overcome the detection of single gel electrophoresis method is analyzed the sheep transferrin.
Background technology
The serum transferrin is that a kind of molecular weight is the iron combination glycoprotein of 75000-80000kDa, and the Zone electophoresis band is positioned at betaglobulin, and major function is that Railway transportation is passed to marrow or tissue storage's organ.Function in animal blood plasma is ferric ion to be transported to the certain position of body from enteron aisle remove synthetic important fortune oxygen instrument; Therefore the trace element that can also combine Cu, Mn, Zn and Co etc. in body growth growth and metabolism, to play an important role is the cell growth and break up the necessary factor.1958, Ashton found the TF polymorphism first in Britain's sheep serum, and the various countries scholar has carried out extensive studies to the TF polymorphism of other kind sheep again subsequently.
Forefathers study more employing starch-gel electrophoresis and come the protein isolate enzyme to this; The method exist resolution lower, might underestimate shortcomings such as hereditary variation level (Sun Wei. the Central Asia is with the research [D] of southeast different ecological type sheep variety Population Genetics. Yangzhou: Yangzhou University, 2006).Research sheep TF polymorphism adopts the polyacrylamide gel vertical slab electrophoresis mostly usually at present.Zhang Caijun (1994) adopts the polyacrylamide gel vertical slab electrophoresis that the serum transferrin polymorphism of 100 Qinghai Yellow Cattle is studied; Find that there are 6 allele in Qinghai Yellow Cattle serum transferrin site; Constitute altogether 13 genotype (Zhang Caijun. Qinghai Yellow Cattle serum transferrin polymorphism research [J]; Qinghai animal and veterinary magazine, 1994,24 (6): 9-11).Liu Yanfen (2002) utilizes the polyacrylamide gel vertical slab electrophoresis that Leizhou goat blood transferrin polymorphism is studied; Find 3 allele altogether, 4 kinds of genotype (Liu Yanfen, Zhao Yuntao; Yan Qitao etc. Leizhou goat blood transferrin polymorphism research [J]; The China plant-eating animal, 2003,5:11-13).Letter gold big (2009) adopts polyacrylamide gel vertical slab electrophoresis method 74 parts of blood samples of Euler's type tibetan sheep to be carried out the research of serum transferrin polymorphism; Find to detect 5 multiple alleles on the serum T F site of seized Euler's type tibetan sheep; 9 kinds of genotype, for the gene pool accumulation basic test data of plateau original inhabitants kind (the letter gold is big. the research [J] of Euler's type tibetan sheep serum transferrin polymorphism, Chinese animal and veterinary; 2010,37 (4): 118-120).Haining, field (2011) is for inquiring into the advantage degree of the different phenotypes of the cold sheep blood serum transferrin of the little tail in Qinghai; Adopt the polyacrylamide gel vertical slab electrophoresis that the cold sheep transferrin polymorphism of 89 little tails introducing Qinghai Province is studied; On the cold sheep transferrin site of little tail to be detected, detect 16 kinds of genotype altogether; Controlled by 7 allele; Show the hereditary variability introducing the cold sheep TF of the little tail in Qinghai and demonstrate abundant bio-diversity and height (Haining, field. cold sheep blood serum transferrin polymorphism research [J] Anhui of the little tail in Qinghai agricultural sciences, 2011,39 (7): 4022-4023).(2010) such as Hai Siyuan, Yang Hulin, Chen Zengguo utilize the polyacrylamide vertical slab electrophoresis to measure Tibetan, meadow, Abbe county, Sichuan Province is the polymorphism of sheep Hongyuan sheep monoid, Jia Luo sheep monoid and Qinghai Euler's sheep blood serum transferrin; Its hereditary feature has been carried out comparative studies; The result shows that Hongyuan sheep transferrin has 3 kinds of genotype; Jia Luoyang has only a kind of genotype, and (polymorphism that .3 Tibetan such as Hai Siyuan, Yang Hulin, Chen Zengguo is sheep variety serum transferrin is [J] relatively; The Sichuan animal and veterinary, 2010,4:30-32).Fan Yuxia (2008) adopts the polyacrylamide gel vertical slab electrophoresis that 82 parts of Qinghai local lowlenthal serum transferrin of helping each other has been carried out electrophoretic analysis; The result finds that seized lowlenthal serum has 3 kinds of genotype; For understanding the help each other TF characteristic of local goat of Qinghai, for the gene pool accumulation experiment material of this kind (Fan Yuxia. the help each other electrophoretic analysis [J] of local lowlenthal serum transferrin and haemoglobin of Qinghai, Sichuan animal and veterinary; 2008,7 (213): 30-32).
The polyacrylamide gel vertical electrophoresis often adopts homogeneous concentration gel, confirms gel strength according to the size of protein molecular weight, and its technology simple operations is convenient; But because gel strength is single, transferrin still can not obtain good somatotype in electrophoresis process, and efficient is still lower comparatively speaking; Offset plate sometimes can generate heat in the experimentation in addition, and the vertical slab electrophoresis process is 60V, 1h, 120V; 2h, the time of need labor.Still do not utilize at present leveling board polyacrylamide gel gradient electrophoresis to detect the report of transferrin polymorphism; The present invention aims to provide a kind of new, efficient, simple, fast detection transferrin genetic polymorphism detection method, for protecting and rationally utilizing local sheep variety resource that referential genetic information is provided.
Summary of the invention
The objective of the invention is to the problem to existing research method existence, for understanding the genetic diversity of sheep transferrin in depth, the leveling board polyacrylamide gel gradient electrophoresis technology that the present invention adopted has then overcome above-mentioned deficiency.The present invention injects the gel of variable concentrations respectively to the bottom from the leveling board top, form gradient; Bigger in aperture, gel top, less at the bottom part aperture diameter of gel, transferrin somatotype in gradient gel is clear, and efficient is high, and the glue process is simple, and adopting voltage earlier is 380V prerunning 10-15 minute, adopts then to get final product in 740V electrophoresis 5-6 hour.
Polyacrylamide gel gradient electrophoresis of the present invention detects the method for transferrin genetic polymorphism; Be to adopt leveling board polyacrylamide gradient gel electrophoresis to detect the transferrin polymorphism; Described leveling board polyacrylamide gradient gel, gel strength is respectively 12%, 8%, 6% to the bottom from the top; Deposition condition is 380V.
In order to reduce the temperature of offset plate in the electrophoresis process, guarantee carrying out smoothly of experiment; Can also carry out ice water circulation cooling (keeping 4 ℃) to leveling board.For reaching this purpose, the temperature of ice water circulation system with offset plate in effective reduction experimentation can be set on electrophoresis equipment.
The step of the inventive method comprises: take the non-anticoagulation of sheep subsequent use; Adopt the horizontal gradient electrophoresis that serum T F is carried out electrophoretic analysis; This process comprises steps such as the preparation, glue, dyeing of preparation, the electrode buffer of gel, and serum T F is carried out polymorphism analysis.
The concrete operations of this method: gather the non-anticoagulation of sheep, make its natural coagulation,, isolate serum, supernatant is placed clean penicillin bottle of sterilization and numbering with the centrifugal 10min of 3000r/min.Water glass cleaning plate, dry, pad be placed on the glass plate,, two blocks of glass plates are put together with that plane that cleansing solution is cleaned the dull and stereotyped no adhesive tape of that piece of having adhesive tape, with clip will around fixing.In glass plate, add 12%, 6%, 8% glue respectively with syringe, treat that glue face level is solidified after, pour out normal butyl alcohol; Take filler strip and glass plate away, on the glue face of residue flat board, place preservative film, prevent that glue from becoming dry; On blank sheet of paper that draws a straight line of offset plate underlay, make the glue lower limb from the about 5.5cm of straight line, each plasma sample bottle to be measured is opened; Get the little scraps of paper of individual layer (2mm*6mm); Be stained with a little blood plasma, draw unnecessary blood plasma, be placed on the following of line and note putting in position standard model with coil paper.
Behind the application of sample offset plate is placed on the flat board of horizontal cyclic device, pours electrode buffer into, make cotton ride over Jiao Mianshang, cotton is close to the glue face.
Deposition condition: prerunning behind the application of sample, voltage 380v, electric current 24ma, 15-20min.
Electrophoretic band stops electrophoresis after running out of about 10cm, keeps 12% part, and the remainder gel is cast out, and gel is entered with the syringe water filling what glue contacted with glass plate on every side, then with expect film rubber cover face, inclination offset plate.Glue is put in the square box that dyeing liquor is housed, and band can appear in a few minutes excessively, forms images through the UVI imaging system, carries out polymorphism analysis this moment.
Beneficial effect of the present invention: the present invention is through leveling board gradient electrophoresis technical Analysis sheep serum transferrin genetic polymorphism; Can be widely used in the analysis of protein genetic polymorphism; Institute's isolated protein wider; And can distinguish that molecular weight differs less protein, for the research of protein genetic polymorphism provides technical support.
The present invention is different from the polyacrylamide gel vertical slab electrophoresis; Its process is not on the gel of single concentration, to carry out; But through forming gradient gel, the concentration in gradient of acrylic amide changes from the top to the bottom to make gel, and is therefore bigger in the aperture, top of gel; And less at the bottom part aperture diameter of gel, transferrin band in this gradient electrophoresis is clear, somatotype efficient high.Characteristics such as that this method has is easy, sensitive, somatotype efficient height; Can analyze sheep serum transferrin genetic polymorphism; For finding and differentiate that more transferrin allele provide detection method, and then reference frame is provided for the research of sheep transferrin genetic polymorphism.
Description of drawings
Each concentration glue length synoptic diagram of Fig. 1.
Fig. 2 embodiment leveling board gradient electrophoresis detects each banding pattern of transferrin (Tf) (allelotype) and declares type figure.
Embodiment
For a better understanding of the present invention, further illustrate content of the present invention below in conjunction with embodiment.
Embodiment 1:
Analyze the step of sheep serum transferrin genetic polymorphism with leveling board polyacrylamide gel gradient electrophoresis:
One, selects to be used to sample to be checked--the non-anticoagulation of-sheep, make its natural coagulation,, isolate serum, supernatant is placed clean penicillin bottle of sterilization and numbering, place-20 ℃ of refrigerators to preserve, supply the TF electrophoresis to use with the centrifugal 10min of 3000r/min.
Two, glue
1. prepare offset plate
Water glass cleaning plate, dry, pad be placed on the glass plate,, two blocks of glass plates are put together with that plane that cleansing solution is cleaned the dull and stereotyped no adhesive tape of that piece of having adhesive tape, with clip will around fixing.
2. add glue
1、12%：
Get 4 big tip heads, put mark A, B, C, DW respectively, get A 11.2ml+B 7.5ml+DW 3.8ml and in a small beaker, get 7.5ml C in above-mentioned small beaker; Glass bar stirs (fast) and draws above-mentioned A, B, C, DW mixed liquor with big syringe, injects offset plate, until 2/3 place of offset plate length; The normal butyl alcohol of little syringe is injected in the glue approximately 1.5ml, approximately 10-20 minute, can see that glue face level solidifies; Outwell top normal butyl alcohol, and water washes the glue face 2 times.
2、6%：
Get A 1.125ml+B 1.5ml+C 1.875ml+DW l.5ml in a small beaker, glass bar stirs and sucks about 6ml with big syringe and inject the glue face, with the about normal butyl alcohol of 1-2ml injection glue face in the little syringe.Treat glue face level, solidify, pour out normal butyl alcohol, water flushing glue face is 1-2 time again.
3、8%：
Get A1.5ml+B1.5ml+C1. 5ml+DW1.5ml in a small beaker, glass bar stirs to get with big syringe approximately gets 6ml, injects the glue face, and remaining normal butyl alcohol injection glue face in the little syringe is left standstill, treat that glue face level is solidified after, pour out normal butyl alcohol.
3. tear glue open
Take filler strip and glass plate away, on the glue face of remaining flat board, place preservative film, on blank sheet of paper that draws a straight line of offset plate underlay, make the glue lower limb from the about 5.5cm of straight line; Each plasma sample bottle to be measured is opened; Get the little scraps of paper of individual layer, be stained with a little blood plasma, draw unnecessary blood plasma with coil paper; Be placed on line below, and note putting in position standard model.
4. electrophoresis process
Behind the application of sample offset plate is placed on the flat board of ice water circulation device, pours electrode buffer into, make cotton ride over Jiao Mianshang, make cotton be close to glue face, smooth.Deposition condition: prerunning behind the application of sample, voltage 380v, electric current 24ma, 15-20 minute.
The electrophoresis circulating device is supporting to have the ice water circulation cooling system.Specific practice is: offset plate is placed on the ice water circulation device flat board above, the flat board through the ice water circulation device comes cooling offset plate (keeping 4 ℃) indirectly.
Three, dyeing
Electrophoretic band stops electrophoresis after running out of about 10cm, keeps 12% part, and the remainder gel is cast out, and gel is entered with the syringe water filling what glue contacted with glass plate on every side, then with expect film rubber cover face, inclination offset plate.Glue is put in the square box that dyeing liquor is housed, and the placement of can spending the night then band can appear, in a few minutes excessively.
Four, declare type
Begin from positive pole, banding pattern is followed successively by A, B, C, D, E, F, G downwards, analyzes banding pattern according to the number of band.
Electrophoretogram is seen Fig. 2
The compound method of the used gel of the present invention:
A. storing solution stock solution:Acrylamide 32g, Bisacrylamide 0.8g, Jia Shui are settled to 100ml
B. gel buffer liquid PH7.8-7.9, Tris 4.54g, TEMD 0.3ml, 2-mercaptoethanol (mercaptoethanol) 0.15ml add water and are settled to 100ml, and use concH
2SO
4Adjust to PH 7.8-7.9.
C. Ammonium persulfate (ammonium persulfate), 100mg/50 ml D.W
Below for making the consumption of a plate glue
(total amount 30) 12%:A.11.2ml+B 7.5ml+C 7.5ml+D.W 3.8ml
(total amount 6) 6%:A.1.125ml+B 1.5ml+C 1.5ml+D.W 1.875ml
(total amount 6) 8%:A.1.5ml+B 1.5ml+C 1.5ml+D.W 1.5ml
Process each the concentration glue length synoptic diagram behind the glue, see Fig. 1
The compound method of electrode used therein damping fluid of the present invention:
Tris 15.74g, boric acid 2.96g add water and are settled to 2000ml, adjust to ph9.0 with NaOH.
The compound method of electrode used therein dyeing liquor of the present invention:
1g Coomassie brilliant blue G is dissolved in the 600ml water, adds the perchloric acid solution of 120ml 6% then, adds water at last till 2000ml.When configuration, adopt the specimen jar of the about 5000ml in laboratory,, therefore can calculate the requirement (x) of 70% perchloric acid, add entry (2000-600-x) then and get final product according to following formula: 120*60%=x*70% because the perchloric acid original liquid concentration is 70%.
Claims (2)
1. a gradient gel electrophoresis detects the method for sheep transferrin polymorphism; It is characterized in that: be to adopt leveling board polyacrylamide gradient gel electrophoresis to detect the transferrin polymorphism; Described leveling board polyacrylamide gradient gel, gel strength is respectively 12%, 8%, 6% to the bottom from the top; Deposition condition is 380V prerunning 10-15 minute for adopting voltage, adopts then 740V electrophoresis 5-6 hour.
2. gradient gel electrophoresis according to claim 1 detects the method for sheep transferrin polymorphism, it is characterized in that in electrophoresis process, leveling board being carried out the ice water circulation cooling, keeps 4 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103675072A (en) * | 2013-11-28 | 2014-03-26 | 南昌大学 | Gradient electrophoresis method for separation and identification of natural protein molecules |
| CN108387630A (en) * | 2018-01-29 | 2018-08-10 | 温州科技职业学院 | A kind of improvement SDS- polyacrylamide gel electrophoresis techniques |
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| US20100252435A1 (en) * | 2006-06-20 | 2010-10-07 | Gerhard Weber | Method and device for separation and depletion of certain proteins and particles using electrophoresis |
| CN102213692A (en) * | 2010-04-01 | 2011-10-12 | 北京师范大学 | Method for detecting proteins |
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2012
- 2012-05-08 CN CN2012101399838A patent/CN102636548A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100252435A1 (en) * | 2006-06-20 | 2010-10-07 | Gerhard Weber | Method and device for separation and depletion of certain proteins and particles using electrophoresis |
| CN102213692A (en) * | 2010-04-01 | 2011-10-12 | 北京师范大学 | Method for detecting proteins |
Non-Patent Citations (3)
| Title |
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| BO GAHNE等: "Horizontal polyacrylamide gradient gel electrophoresis for the simultaneous phenotyping of transferring, post-transferrin, albumin and post-albumin in the blood plasma of cattle", 《ANIM. BLOOD GRPS BIOCHEM. GENET.》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675072A (en) * | 2013-11-28 | 2014-03-26 | 南昌大学 | Gradient electrophoresis method for separation and identification of natural protein molecules |
| CN103675072B (en) * | 2013-11-28 | 2017-01-04 | 南昌大学 | A kind of gradient electrophoresis method for native protein molecule isolation identification |
| CN108387630A (en) * | 2018-01-29 | 2018-08-10 | 温州科技职业学院 | A kind of improvement SDS- polyacrylamide gel electrophoresis techniques |
| CN108387630B (en) * | 2018-01-29 | 2019-09-03 | 温州科技职业学院 | A kind of improvement SDS- polyacrylamide gel electrophoresis technique |
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Application publication date: 20120815 |