CN103728171B - Extraction method of earthworm total protein suitable for two-dimensional electrophoresis experiment - Google Patents
Extraction method of earthworm total protein suitable for two-dimensional electrophoresis experiment Download PDFInfo
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Abstract
The invention discloses an extraction method of earthworm total protein suitable for two-dimensional electrophoresis experiment. The extraction method comprises the following steps: grinding fresh or -80 DEG C stored earthworm into earthworm powder with liquid nitrogen, adding a protein extraction solution into the earthworm powder, carrying out vortex blending on the mixture and precipitating the mixture solution; centrifuging the mixture solution, removing the supernatant, adding dithiothreitol acetone solution to wash the precipitates, centrifuging; adding dithiothreitol solution to wash the precipitates, centrifuging, allowing the liquid to vaporize; dissolving the precipitates in a protein lysis buffer, carrying out vortex blending on the mixture, allowing the mixture to stand still in an ice bath; adding dithiothreitol into the mixture, carrying out vortex blending on the mixture, and allowing the mixture to stand still in the ice bath; carrying out ultrasound treatment and centrifuging treatment on the mixture in the ice bath to obtain the supernatant and subpackaging the supernatant at -80 DEG C. The method is less in operation steps and time consumption and easy to operate, and moreover makes up the defects that the impurities in the earthworm samples such as lipids, polysaccharides and phenols cannot be effectively removed by the simple precipitation method. By adopting the method, the protein points in the two-dimensional electrophoresis spectrogram can be distributed in a wide isoelectric point and molecular weight range.
Description
Technical field
The present invention relates to a kind of extracting method of the earthworm total protein being applied to dielectrophoresis experiment.
Background technology
The preparation of protein sample occupies very important status in the middle of whole proteomics research, is also to determine experiment
The key factor of success or failure, the quality of sample quality, directly determine the result of downstream experiment.Preferably protein sample preparation method
Should be to dissolve all protein to greatest extent, adopt minimum operating procedure as far as possible, avoid as far as possible protein loss,
Degraded and change, reduce the pollution to protein for the impurity as far as possible, and have preferable repeatability.
For the extraction of earthworm total protein, difficulty is that earthworm total protein is to be extracted with whole piece individuality, therefore its body
Metabolite or the secondary metabolites such as interior lipid, polysaccharide and phenols, and multiple oxidizing ferment and protease, all can be one
Determine to have influence on purity and the quality of total protein extraction in degree.
Content of the invention
It is an object of the invention to provide a kind of extraction of the high earthworm total protein being applied to dielectrophoresis experiment of recovery rate
Method.
Technical scheme is summarized as follows：
A kind of extracting method of the earthworm total protein being applied to dielectrophoresis experiment, comprises the steps：
1) fresh or -80 DEG C of preservations earthworms are taken to become earthworm powder with liquid nitrogen grinding in the mortar of precooling；
2) 1g earthworm powder is taken to add in 20ml protein extract, vortex 2-6 minute mixes, little in -20 DEG C of precipitation 6-16
When；Centrifugation, abandons supernatant, adds the acetone soln washing precipitation that 10-30ml mass concentration is 0.3% dithiothreitol (DTT), after washing
In 4 DEG C, it is centrifuged 30 minutes, repeat washing, centrifugally operated 2 times；
3) the dithiothreitol (DTT) solution being 0.3% with mass concentration washs precipitation, under the centrifugal condition of 12000 × g, centrifugation
30 minutes, abandon supernatant, volatilize remaining liquid in precipitation, the solvent of described dithiothreitol (DTT) solution is 70%- for volumetric concentration
90% aqueous acetone solution；
4) take what 0.1-0.3g step (3) obtained to be precipitated and dissolved in 1ml protein lysate, be vortexed and mix, ice bath standing 5
Minute；Add dithiothreitol (DTT) to make its final concentration of 40-100mM, be vortexed and mix, stand 10 minutes on ice；
5) under ice bath, with 250-350W power, the ultrasonic 25-35 circulation of 2s/6s, 13500 × g is centrifuged 40 minutes, takes
- 80 DEG C clearly packing preserve；
Described protein extract is made with following methods：10-30g trichloroacetic acid and 0.3g dithiothreitol (DTT) is taken to add acetone extremely
100ml dissolves；
Described protein lysate is made with following methods：Take 4.2g urea, 1.52g thiocarbamide and 0.2g3- [(3- cholesterol ammonia
Propyl group) dimethylamino] -1- propane sulfonic acid deionized water is settled to 10ml.
Preferably protein extract is made with following methods：20g trichloroacetic acid and 0.3g dithiothreitol (DTT) is taken to add acetone extremely
100ml dissolves.
Step (3) is preferably：The dithiothreitol (DTT) solution being 0.3% with mass concentration washs precipitation, 12000 × g from
Under the conditions of the heart, it is centrifuged 30 minutes, the aqueous acetone solution that the solvent of described dithiothreitol (DTT) solution is 80% for volumetric concentration；Volatilize
Liquid.
Step (4) is preferably：Take what 0.1-0.3g step (3) obtained to be precipitated and dissolved in 1ml protein lysate, be vortexed mixed
Even, ice bath stands 5 minutes；Add dithiothreitol (DTT) to make its final concentration of 65mM, be vortexed and mix, stand 10 minutes on ice.
Step (5) is preferably：Under ice bath, with 300W power, ultrasonic 30 circulations of 2s/6s, 13500 × g is centrifuged 40 minutes,
- 80 DEG C of packing of supernatant are taken to preserve.
Advantage：
The method of the present invention is few in operating procedure, take short, simple on the basis of, compensate for single solvent precipitate not
The deficiency of the impurity such as lipid in the middle of earthworm sample, polysaccharide and phenols can effectively be removed.It is two-way that the method process obtains
In electrophoretic image, protein site can be distributed in wide in range an isoelectric point and molecular weight ranges.
Brief description
Fig. 1 is the electrophoretogram of the earthworm total protein that embodiment 1 is extracted.
Fig. 2 is the electrophoretogram of the earthworm total protein that embodiment 2 is extracted.
Fig. 3 is the electrophoretogram of the earthworm total protein that embodiment 3 is extracted.
Fig. 4 is the electrophoretogram of the earthworm total protein that comparative example 1 is extracted.
Specific embodiment
Following case facilitates a better understanding of the present invention, the experimental technique in case study on implementation, is often if no special instructions
Rule operation.Reagent material used in subordinate's case study on implementation, if no special instructions, is purchased from routine biochemistry Reagent Company.Below
Quantitative test in case study on implementation, is respectively provided with three times and repeats to test, results averaged.
Urea：Sigma Co., USA dispenses, article No.：U0631.Thiocarbamide：Sigma Co., USA dispenses, article No.：T7578.
3- [3- (courage amido propyl) dimethylamino] propane sulfonic acid inner salt：Purchased from Sigma Co., USA, article No.：C9426.Dithiothreitol (DTT),
Purchased from Sigma Co., USA, article No.：D9163.Trichloroacetic acid, purchased from Sigma Co., USA, article No.：T9159.Bio-Lyte carries
Body ampholytes, purchased from Bio-Rad company of the U.S., article No.：163-1113.Compatriot's love victory (Eisenia used by test
Fetida) earthworm, purchased from Tianjin Jialiming Earthworm Breeding Co., Ltd..
Embodiment 1
First, a kind of extracting method of the earthworm total protein being applied to dielectrophoresis experiment, comprises the steps：
(1) 3 parallel test, takes the compatriot of the fresh health with ripe annulus to like victory earthworm (every earthworm weight
300-600mg) in the mortar of precooling, become powder with liquid nitrogen grinding；
(2) 1g earthworm powder is taken to add in 20ml protein extract, (prepared by protein extract：Take 20g trichloroacetic acid and
0.3g dithiothreitol (DTT) adds acetone and dissolves to 100ml), it is vortexed 4 minutes and mixes, precipitate 10 hours in -20 DEG C；12000 × g is centrifuged
30min, abandons supernatant, adds the acetone soln washing precipitation that 20ml mass concentration is 0.3% dithiothreitol (DTT), washs after 4 DEG C,
Centrifugation 30 minutes, repeats washing, centrifugally operated 2 times；
(3) the dithiothreitol (DTT) solution being 0.3% with mass concentration washs precipitation, under the centrifugal condition of 12000 × g, from
The heart 30 minutes, abandons supernatant, volatilizes remaining liquid in precipitation, and the solvent of described dithiothreitol (DTT) solution is 80% for volumetric concentration
Aqueous acetone solution；
(4) take what 0.1g step (3) obtained to be precipitated and dissolved in 1ml protein lysate, be vortexed and mix, ice bath stands 5 points
Clock；Add dithiothreitol (DTT) to make its final concentration of 65mM, be vortexed and mix, stand 10 minutes on ice；
(5) under ice bath, with 300W power, ultrasonic 30 circulations of 2s/6s, 13500 × g is centrifuged 40 minutes, takes -80 DEG C of supernatant
Packing preserves.
Protein lysate is made with following methods：Take 4.2g urea, 1.52g thiocarbamide and 0.2g3- [(3- cholesterol ammonia third
Base) dimethylamino] -1- propane sulfonic acid deionized water is settled to 10ml.
2nd, extraction effect identification
1st, determination of protein concentration
Using classical Bradford method, that is, Coomassie brilliant G-250 sizing technique is to albumen in the supernatant of above-mentioned preparation
The concentration of matter is measured.Recording protein concentration is 9.23mg/ml.
2nd, isoelectric focusing electrophoresis
(1) take out -20 DEG C of IPG adhesive tape deposited (purchased from Bio-Rad company of the U.S.), place 10min in room temperature.
(2) supernatant preparing embodiment 1 step one, adds the dithiothreitol (DTT) of 0.3g and the Bio-lyte of 2.5 μ L,
And 1 μ L bromophenol blue, be configured to protein content be 1mg volume be 300 μ L sample solution.
(3) along focusing on plate edge LINEAR CONTINUOUS addition sample solution, remove the protective layer in IPG adhesive tape with tweezers afterwards.
(4) IPG adhesive tape glue surface is slowly covered downwards above sample solution, it is to avoid produce bubble.
(5), after 30min, 2-3mL mineral oil is covered on adhesive tape.
(6) to good both positive and negative polarity, close the lid, isoelectric focusing program is set according to table 1 and carries out isoelectric focusing electrophoresis.
Table 117cm adhesive tape isoelectric focusing program is arranged
3rd, the second to SDS-PAGE electrophoresis
(1) prepare the acrylamide gel solution that 40ml mass concentration is 12.5%, in implantation glass plate interlayer, top is stayed
The space of 1cm, with MilliQ water seal face, keeps glue surface smooth, is polymerized 40 minutes, gel is layered with upper liquid.
(2) after gel sets, remove the MilliQ water on separation gel surface, rinsed with MilliQ water.
(3) first place dry thick filter paper on the table, the adhesive tape glue surface that isoelectric focusing electrophoresis is focused on is placed on dry upward
On thick filter paper.By another thick filter paper MilliQ water-soaked, squeeze and remove excessive moisture, be then placed directly within adhesive tape, gently inhale
Mineral oil on immobilized ph gradient strip and redundant sample.Adhesive tape is transferred in aquation disk, groove adds 6ml adhesive tape level pad I.
Aquation disk is placed on horizontal shaker and slowly rocks 15 minutes.After balance terminates for the first time, thoroughly outwell or sop up in aquation disk
Adhesive tape level pad I.And draw unnecessary adhesive tape level pad I with filter paper.Add adhesive tape level pad II, continue
Continue slowly to rock on horizontal shaker 15 minutes and carry out second balance.
Adhesive tape level pad mother liquor is made with following methods：36g urea, 2g SDS, 25ml concentration is taken to be 1.5mol/L
PH be 8.8 Tris-HCl buffer solution and 20ml glycerine add water and be settled to 100ml, the every pipe 10ml of packing, -20 DEG C of refrigerators are protected
Deposit.
Adhesive tape level pad I preparation method is：0.20g bis- sulphur threose is added in 10mL adhesive tape level pad mother liquor
Alcohol.
Adhesive tape level pad II preparation method is：0.25g iodoacetamide is added in 10mL adhesive tape level pad mother liquor
(keeping in Dark Place).
(4) suck unnecessary between glass plate liquid above the polyacrylamide gel that step (2) obtains with filter paper.By glass
Plate is put on the table, long glass plate under, upward, the top of gel is against oneself for short glass plate.
(5) low melting-point agarose sealing liquid is carried out heating for dissolving.
(6) by 10 × electrophoretic buffer, dilute 10 times with graduated cylinder, make 1 × electrophoretic buffer.
(7) the adhesive tape level pad II in aquation disk after second balances for the step (3) is thoroughly outwelled or
Sopped up by filter paper.
(8) IPG adhesive tape is removed from sample hydration disk, make glue surface soak end completely 1 with one end that adhesive tape clamped by tweezers
In × electrophoretic buffer.Then adhesive tape glue surface is placed on the long glass plate of gel upward.
(9) PAGE gel being placed with adhesive tape is transferred on encapsulating frame, short glass plate one is facing to oneself.In gel
Top add low melting-point agarose sealing liquid.
(10) use tweezers, spatula or the syringe needle of tack, lightly adhesive tape is pushed down on, be allowed to coagulate with polyacrylamide
Glue glue surface completely attaches to.It is careful not to below adhesive tape produce any bubble.Pushing away adhesive tape with tweezers, spatula or tack syringe needle
When it should be noted that be promote the gel back side support membrane, glue surface must not be encountered.Place 5 minutes, make low melting-point agarose sealing liquid thorough
Bottom solidifies.
(11) after low melting-point agarose sealing liquid solidifies completely.Gel is transferred in electrophoresis tank, carries out electrophoresis (electrophoresis
Program is set to 2W, 25min；15W is to terminating).
(12) after electrophoresis terminates, gently pry open layer glass, take out gel, carry out coomassie brilliant blue staining.
The preparation method of described low melting-point agarose sealing liquid is：By 0.5g low melting-point agarose, 0.303g Tris alkali
(purchased from Bio-Rad company of the U.S.), 1.44g glycine (purchased from Bio-Rad company of the U.S.), 1ml mass fraction is 10% SDS
The bromophenol blue solution being 1% with 100 μ l mass fractions, plus MilliQ water is settled to 100ml, heating for dissolving to clarifying, protect by room temperature
Deposit.
The preparation method of described 10 × electrophoretic buffer is：By the Tris alkali of 30g, 144g glycine and 10g SDS (are purchased from
Bio-Rad company of the U.S.), plus MilliQ water is settled to 1L, after mixing, room temperature preservation.
Result is shown in Fig. 1.
Embodiment 2
First, a kind of extracting method of the earthworm total protein being applied to dielectrophoresis experiment
(1) 3 parallel test, takes the compatriot of the fresh health with ripe annulus to like victory earthworm (every earthworm weight
300-600mg) in the mortar of precooling, become powder with liquid nitrogen grinding；
(2) 1g earthworm powder is taken to add in 20ml protein extract, (the preparation of this protein extract：Take 10g trichloroacetic acid
Add acetone with 0.3g dithiothreitol (DTT) to dissolve to 100ml), it is vortexed 2 minutes and mixes, precipitate 16 hours in -20 DEG C；12000 × g from
Heart 30min, abandons supernatant, adds the acetone soln washing precipitation that 10ml mass concentration is 0.3% dithiothreitol (DTT), washs after 4
DEG C, it is centrifuged 30 minutes, repeat washing, centrifugally operated 2 times；
(3) the dithiothreitol (DTT) solution being 0.3% with mass concentration again washs precipitation, under the centrifugal condition of 12000 × g,
Centrifugation 30 minutes, abandons supernatant, volatilizes remaining liquid in precipitation, and the solvent of described dithiothreitol (DTT) solution for volumetric concentration is
70% aqueous acetone solution；
(4) take what 0.1g step (3) obtained to be precipitated and dissolved in 1ml protein lysate, be vortexed and mix, ice bath stands 5 points
Clock；Add dithiothreitol (DTT) to make its final concentration of 40mM, be vortexed and mix, stand 10 minutes on ice；
(5) under ice bath, with 250W power, ultrasonic 25 circulations of 2s/6s, 13500 × g is centrifuged 40 minutes, takes -80 DEG C of supernatant
Packing preserves.
The preparation of protein lysate is with embodiment 1.
2nd, extraction effect identification
Step 2 with embodiment 1
Result is shown in Fig. 2.
Embodiment 3
First, a kind of extracting method of the earthworm total protein being applied to dielectrophoresis experiment
(1) 3 parallel test, takes compatriot's love victory earthworm (every earthworm weight 300-600mg) of the health of -80 DEG C of preservations
Become powder with liquid nitrogen grinding in the mortar of precooling；
(2) 1g earthworm powder is taken to add in 20ml protein extract, (prepared by protein extract：Take 30g trichloroacetic acid and
0.3g dithiothreitol (DTT) adds acetone and dissolves to 100ml) being vortexed 6 minutes mixes, precipitate 6 hours in -20 DEG C；12000 × g is centrifuged
30min, abandons supernatant, adds the acetone soln washing precipitation that 30ml mass concentration is 0.3% dithiothreitol (DTT), washs after 4 DEG C,
Centrifugation 30 minutes, repeats washing, centrifugally operated 2 times；
(3) the dithiothreitol (DTT) solution being 0.3% with mass concentration cleans precipitation, under the centrifugal condition of 12000 × g, from
The heart 30 minutes, abandons supernatant, volatilizes remaining liquid in precipitation, and the solvent of described dithiothreitol (DTT) solution is 90% for volumetric concentration
Aqueous acetone solution；
(4) take what 0.3g step (3) obtained to be precipitated and dissolved in 1ml protein lysate, be vortexed and mix, ice bath stands 5 points
Clock；Add dithiothreitol (DTT) to make its final concentration of 100mM, be vortexed and mix, stand 10 minutes on ice；
(5) under ice bath, with 350W power, ultrasonic 35 circulations of 2s/6s, 13500 × g is centrifuged 40 minutes, takes -80 DEG C of supernatant
Packing preserves.
Protein lysate is prepared with embodiment 1.
2nd, extraction effect identification
Step 2 with embodiment 1
Result is shown in Fig. 3.
Comparison example 1
First, the extraction of earthworm total protein
The compatriot taking the health with ripe annulus likes that victory earthworm (every earthworm weight 300-600mg) three carries out total egg
White extraction, comprises the following steps that：
(1) 3 parallel test, takes the compatriot of the fresh health with ripe annulus to like that victory earthworm is used in the mortar of precooling
Liquid nitrogen grinding becomes powder；
(2) 1g earthworm powder is taken to add in 20ml protein extract, (prepared by protein extract：Take 20g trichloroacetic acid and
0.3g dithiothreitol (DTT) adds acetone and dissolves to 100ml) being vortexed 4 minutes mixes, precipitate 10 hours in -20 DEG C；12000 × g is centrifuged
30min, abandons supernatant, adds the acetone soln washing precipitation that 20ml mass concentration is 0.3% dithiothreitol (DTT), washs after 4 DEG C,
Centrifugation 30 minutes, repeats washing, centrifugally operated 3 times, volatilizes liquid；
(3) take what 0.1g step (2) obtained to be precipitated and dissolved in 1ml protein lysate, be vortexed and mix, ice bath stands 5 points
Clock；Add dithiothreitol (DTT) to make its final concentration of 65mM, be vortexed and mix, stand 10 minutes on ice；
(4) in 4 DEG C, 13500 × g is centrifuged 40min, takes -80 DEG C of packing of supernatant to preserve.
Described protein extract and the preparation method of protein lysate：With embodiment 1
2nd, extraction effect identification
Step 2 with embodiment 1
Result is shown in Fig. 4.
The method that comparative example is adopted, with reference to from literary composition
Offer:Wang,X.；Chang,L.；Sun,Z.；Zhang,Y.；Yao,L.,Analysis of earthworm
Eisenia fetida proteomes during cadmium exposure:An ecotoxicoproteomics
approach.Proteomics.2010,10,(24),4476-90.
Result explanation：By carrying out Experimental Comparison with the earthworm total protein extraction method delivered in the recent period, by dielectrophoresis egg
White matter expresses the species that spectrogram can be seen that the earthworm total protein of method of the present invention extraction, i.e. point on dielectrophoresis glue figure
Widely distributed property, control methods to be more than, the removal effect of high-abundance proteins is more preferable simultaneously.I.e. the method for the present invention extracts earthworm
The quality of earthworm total protein, higher than presentation method, is to carry out earthworm holoprotein group credit using bidirectional electrophoresis technique to analyse and follow-up
Test provides technical guarantee.
Claims (4)
1. a kind of extracting method of the earthworm total protein being applied to dielectrophoresis experiment, is characterized in that comprising the steps：
1) fresh or -80 DEG C of preservations earthworms are taken to become earthworm powder with liquid nitrogen grinding in the mortar of precooling；
2) 1g earthworm powder is taken to add in 20ml protein extract, vortex 2-6 minute mixes, and precipitates 6-16 hour in -20 DEG C；From
The heart, abandons supernatant, adds the acetone soln washing precipitation that 10-30ml mass concentration is 0.3% dithiothreitol (DTT), washs after 4 DEG C,
Centrifugation 30 minutes, repeats washing, centrifugally operated 2 times；
3) the dithiothreitol (DTT) solution being 0.3% with mass concentration washs precipitation, under the centrifugal condition of 12000 × g, is centrifuged 30 points
Clock, abandons supernatant, volatilizes remaining liquid in precipitation, and the solvent of described dithiothreitol (DTT) solution is 70%-90% for volumetric concentration
Aqueous acetone solution；
4) take 0.1-0.3g step 3) being precipitated and dissolved in 1ml protein lysate of obtaining, it is vortexed and mixes, ice bath stands 5 minutes；
Add dithiothreitol (DTT) to make its final concentration of 40-100mM, be vortexed and mix, stand 10 minutes on ice；
5) under ice bath, with 250-350W power, the ultrasonic 25-35 circulation of 2s/6s, 13500 × g is centrifuged 40 minutes, takes supernatant -80
DEG C packing preserve；
Described protein extract is made with following methods：Take 20g trichloroacetic acid and 0.3g dithiothreitol (DTT) to add acetone molten to 100ml
Solution；
Described protein lysate is made with following methods：Take 4.2g urea, 1.52g thiocarbamide and 0.2g3- [(3- cholesterol ammonia third
Base) dimethylamino] -1- propane sulfonic acid deionized water is settled to 10ml.
2. extracting method according to claim 1 is it is characterised in that described step 3) be：It is 0.3% with mass concentration
Dithiothreitol (DTT) solution washing precipitation, under the centrifugal condition of 12000 × g, be centrifuged 30 minutes, described dithiothreitol (DTT) solution molten
The aqueous acetone solution that agent is 80% for volumetric concentration；Volatilize liquid.
3. extracting method according to claim 1 is it is characterised in that described step 4) be：Take 0.1g step 3) obtain heavy
Shallow lake is dissolved in 1ml protein lysate, is vortexed and mixes, and ice bath stands 5 minutes；Dithiothreitol (DTT) is added to make it final concentration of
65mM, is vortexed and mixes, and stands 10 minutes on ice.
4. extracting method according to claim 1 is it is characterised in that described step 5) be：Under ice bath, with 300W power,
Ultrasonic 30 circulations of 2s/6s, 13500 × g is centrifuged 40 minutes, takes -80 DEG C of packing of supernatant to preserve.
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| CN104075924B (en) * | 2014-07-15 | 2017-02-08 | 中国农业科学院农产品加工研究所 | Preparation and dielectrophoresis technology suitable for total protein of skeletal muscle of ruminants |
| CN104596826B (en) * | 2015-01-14 | 2018-01-19 | 王共先 | The extracting method of albumen in a kind of stone in urinary system |
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