CN106632582A - Extraction method of soil protein - Google Patents
Extraction method of soil protein Download PDFInfo
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- CN106632582A CN106632582A CN201710068838.8A CN201710068838A CN106632582A CN 106632582 A CN106632582 A CN 106632582A CN 201710068838 A CN201710068838 A CN 201710068838A CN 106632582 A CN106632582 A CN 106632582A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention provides an extraction method of soil protein. The extraction method comprises the following steps of adding soil protein extraction buffer liquid into a soil sample, oscillating, and standing; putting into boiling water for heating, cooling in 0 DEG C ice water, oscillating, and centrifuging; filtering supernatant, adding Tris saturated phenol into the filtered supernatant, oscillating, centrifuging, removing the supernatant, and collecting lower phenol solution; adding an ammonium acetate methanol solution into the lower phenol solution, and storing for 6 to 8h at the temperature of -20 DEG C; centrifuging, removing out the supernatant, collecting protein precipitate, cleaning the protein precipitate by acetone, and freeze-drying, so as to obtain protein dry powder. After detecting by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) electrophoresis, compared with the protein band obtained by other two representative extraction methods of the soil protein, the band of the soil protein extracted by the method is clearer.
Description
Technical field
The present invention relates to a kind of soil method for extracting protein.
Background technology
Environment Metaproteomics are a kind of investigative techniques of rising in recent years, and the technology can will be complicated micro- in soil
Biocenose effectively carries out comprehensive analysis, to disclose soil ecosystem in change.
Metaproteomics studying a question in soil environment it is critical that the extraction and analysis of soil protein, its
The extraction of middle protein is the emphasis for studying soil environment.Before the extraction soil proteomics research of soil protein
Carry, yet with the shortage of soil protein extracting method, the research of current soil proteomics can't effectively be carried out.
Therefore, the present invention is optimized for the key technology in terms of the grand Protein Extraction of soil, can be in the grand egg of soil to the present invention
Certain contribution is made in the research of white matter group.
The content of the invention
It is an object of the invention to provide a kind of effective soil method for extracting protein.
Soil method for extracting protein provided by the present invention, comprises the steps：
1) soil protein extraction buffer, concussion is added to stand in pedotheque；
2) it is placed in boiling water and heats, cools down in 0 DEG C of frozen water, concussion, centrifugation；
3) by supernatant liquid filtering, Tris saturated phenols, concussion is added to be centrifuged, abandoning supernatant in filtered supernatant,
Collect lower floor's phenol solution；
4) methyl alcohol ammonium acetate solution, -20 DEG C of placement 6h-8h are added in lower floor's phenol solution；
5) it is centrifuged, supernatant is removed, collect protein precipitation, protein precipitation is cleaned with acetone, freeze, obtains egg
White matter dry powder.
Said method step 1) in, the soil protein extraction buffer is neutral protein extraction buffer, is had
Body can be the protein extraction buffer of pH6.8.
The compound method of the protein extraction buffer of the pH6.8 is：Lauryl sodium sulfate (SDS) 1.25g is weighed,
Trishydroxymethylaminomethane (Tris) 1.2114g, NaCl 0.8766g, dithiothreitol (DTT) (DTT) 0.386g adds the double steamings of 70ml
Water dissolves, using concentrated hydrochloric acid pH to pH 6.8 is adjusted, and is finally settled to 100ml using distilled water, you can.
The pedotheque is 1.8g-2.2g with the proportioning of soil protein extraction buffer：5ml.
The speed of the concussion is 3000rpm-3200rpm, and the time is 10-20min, concretely 15min.
The time of the standing is 3-5min, concretely 3min.
Said method step 2) in, the time of the heating is 5-15min, concretely 10min.
The time of the cooling is 3-5min, concretely 3min.
The speed of the concussion is 3000rpm-3200rpm, and the time is 5-10min, concretely 5min.
The condition of the centrifugation is：4500-5500rpm centrifugations 5-10min at 4 DEG C, concretely at 4 DEG C 5000rpm from
Heart 5min.
Said method step 3) in, it is described to filter using 0.45 μm of nylon leaching film.
The pH value of the Tris saturated phenols is 8.0.
The supernatant is 3-4ml with the proportioning of Tris saturated phenols：2ml.
Tris saturated phenols are purchased from Beijing Suo Laibao scientific ＆ technical corporation.
The speed of the concussion is 3000rpm-3200rpm, and the time is 15-30min, concretely 20min.
The condition of the centrifugation is：10000-13000rpm is centrifuged 15-20min at 4 DEG C, concretely 4 DEG C of 12000rpm
Centrifugation 20min.
Said method step 4) in, the concentration of the methyl alcohol ammonium acetate solution is 0.1M.
The compound method of the methyl alcohol ammonium acetate solution is：Weigh 0.7708g ammonium acetates to be dissolved in 80ml methanol solutions, and
It is settled to 100ml.
The methyl alcohol ammonium acetate solution is 5-6 with the volume ratio of phenol solution：1.
Said method step 5) in, the condition of the centrifugation is：13000-15000rpm centrifugations 15-20min, tool at 4 DEG C
Body can be centrifuged 20min for 4 DEG C of 15000rpm.
The acetone is the acetone of precooling.
The cleaning can be carried out repeatedly, concretely 3 times.
The lyophilized temperature is -80 DEG C.
Jing SDS-PAGE electrophoresis detections find the more other two kinds of tools of the band of the soil protein extracted by said method
Representational soil protein extracting method (Wang et al., 2011 soil protein extracting methods, concrete grammar reference
Paper：Characterization of Metaproteomics in Crop Rhizospheric Soil.Journal of
Proteome Research, 2011 (10)：932-940；Chourey et al., 2010 soil protein extracting methods, specifically
Method reference papers：Direct Cellular Lysis/Protein Extraction Protocol for Soil
Metaproteomics.Journal of Proteome Research, 2010 (9)：6615-6622.) the protein for obtaining
Band is apparent.
Wherein, Separation of Proteins detection method is：Weigh the lyophilized protein powders of 1.5mg and add 1ml SDT buffer solutions
Dissolving obtains protein solution, takes 20 μ l protein solutions in 2ml centrifuge tubes, adds 5 μ 5 × sample-loading buffers of l to be prepared into
Protein example, be vortexed boiling water bath heating 10min after mixing, and treats that sample is cooled down, and taking 20 μ l protein examples carries out SDS-PAGE
Electrophoresis, gel specification is 12 × 16 × 18cm, and protein example adds electrophoretic buffer in electrophoresis tank, and concentration glue is poly- for 5%
Acrylamide gel, 80V, electrophoresis 30min, separation gel is 12% polyacrylamide gel, and 120V, electrophoresis 90min, 15mA is permanent
Determining electric current carries out electrophoresis, and room temperature after electrophoresis terminates, unloads film, and coomassie brilliant blue staining, destainer decolourizes.
It is an advantage of the current invention that：
Content of humic acid is high in soil, and wherein humic acid HA is dissolved in aqueous slkali, and fulvic acid FA had both been dissolved in alkali, also be soluble in water and
Acid, humin HM is dissolved in diluted alkaline, water insoluble and acid, it can thus be appreciated that the pH of protein extraction buffer is to extracting protein influence
It is larger, so neutral pH protein extract buffer can farthest mitigate the impact of humic acid, and in extraction process, boiling
Heating water bath can help cell effectively to crack and inactivate protease, prevent protein degradation.The present invention is by neutral egg
It is dry that white matter Extraction buffer and boiling water bath add the soil Protein Extraction method of thermal farthest to eliminate humic acid
Disturb.
For the more other two kinds of representative soil protein extracting methods of black soil protein for being extracted, Jing
Band is clear after SDS-PAGE detections.
Description of the drawings
Fig. 1 is Wang et al., the soil albumen that 2011, Chourey et al., 2010 and the inventive method are extracted
The SDS-PAGE glue figures of matter extracting method.Wherein, band 1 is using Wang et al., the tomato rhizosphere of 2010 method extraction
Soil protein；Band 2 adopts Chourey et al., the tomato rhizosphere soil protein of 2010 method extraction；Band 3 is adopted
The tomato rhizosphere soil protein extracted with the inventive method.
Fig. 2 is the SDS-PAGE glue figures that this method extracts different soils protein.Wherein, band 4 is using present invention side
The protein of the open ground soil that method is extracted；Band 5 is the protein of the tomato Rhizosphere Soil extracted using the inventive method；Band 6
It is the tomato Rhizosphere Soil after the wheat stalk returning extracted using the inventive method.
Specific embodiment
Below by specific embodiment, the present invention will be described, but the invention is not limited in this.
Experimental technique used in following embodiments if no special instructions, is conventional method；Institute in following embodiments
Reagent, material etc., if no special instructions, commercially obtain.
Tris saturated phenols used in following embodiments are purchased from Beijing Suo Laibao scientific ＆ technical corporation.
The compound method of the reagent used in following embodiments is：
The compound method of the protein extraction buffer of 100ml：Weigh SDS 1.25g, Tris 1.2114g, NaCl
0.8766g, dithiothreitol (DTT) (DTT) 0.386g, add the dissolving of 70ml distilled waters, adjust pH to pH 6.8 using concentrated hydrochloric acid, most
It is settled to 100ml using distilled water afterwards.
The preparation of 100ml 0.1M methyl alcohol ammonium acetate solutions：Weigh 0.7708g ammonium acetates to be dissolved in 80ml methanol solutions, and
It is settled to 100ml.
10ml SDT buffers：SDS 0.4g, Tris 0.121g, DTT 0.039g, in being dissolved in 5ml distilled waters,
PH to pH 6.8 is adjusted using concentrated hydrochloric acid, finally 10ml. is settled to using distilled water
The concentration glue of 5ml 5% is prepared：Distilled water 3.4ml, 30% acrylamide mixed liquor 0.83ml (29.2g acryloyls
Amine is dissolved in 50ml distilled waters, adds 0.8g bisacrylamides, and using distilled water 100ml is settled to, and is filtered, 4 DEG C of preservations),
1.0M Tris-HCl pH6.8,0.63ml (weigh 12.114g to be dissolved in 70ml distilled waters, pH adjusted to 6.8 using concentrated hydrochloric acid,
100ml, 4 DEG C of preservations are being settled to distilled water), 10% ammonium persulfate 50ul (weighs 0.1g ammonium persulfates and is placed in 1.5ml centrifugations
Guan Zhong, adds 1ml distilled waters, maximal rate to be vortexed and mix, matching while using), 10%SDS solution 50ul (weigh 10g SDS additions
80ml distilled waters, the heating for dissolving in 37 DEG C of thermostat water baths, using distilled water 100ml is settled to), tetramethylethylenediamine
(TEMED)5ul。
The separation gel of 10ml 12% is prepared：Distilled water 3.3ml, 30% acrylamide mixed liquor 4ml, 1.5M Tris-HCl
PH8.8,2.5ml (weigh 18.15g to be dissolved in 70ml distilled waters, using concentrated hydrochloric acid pH to 8.8 are adjusted, be settled to distilled water
100ml, 4 DEG C of preservations), 10% ammonium persulfate 100ul, 10%SDS solution 100ul, tetramethylethylenediamine (TEMED) 4ul.
5 times of Tris- glycine buffers are prepared：94g glycine is dissolved in 500ml distilled waters, and 15.1g is added after dissolving
Tris and 5g SDS, treat whole dissolvings, and using distilled water constant volume 1L is stopped, and are stored in 4 DEG C.50ml buffer solutions are measured when using.Use
Distilled water is diluted to 500ml.
Coomassie brilliant blue staining liquid is prepared：Weigh 1.25g coomassie brilliant blue R_250s to be dissolved in 250ml methyl alcohol, add a small amount of
80ml glacial acetic acids are added after distilled water, using distilled water constant volume 1000ml, rapid filtration are stopped.
Destainer is prepared：250ml methyl alcohol adds 80ml glacial acetic acids to be settled to 1L using distilled water.
Wang et al., 2011 soil protein extracting methods, concrete grammar reference papers：Characterization
Of Metaproteomics in Crop Rhizospheric Soil.Journal of Proteome Research, 2011
(10)：932-940.
Chourey et al., 2010 soil protein extracting methods, concrete grammar reference papers：Direct
Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics.Journal of
Proteome Research, 2010 (9)：6615-6622.
Embodiment
1 soil Protein Extraction step：
The extraction of soil protein：Weigh respectively after 2g terrestrial soils, 2g tomato rhizosphere soils, 2g wheat stalk returnings kind
Eggplant Rhizosphere Soil, 3 kinds of soil are respectively placed in 10ml centrifuge tubes, add 5ml soil protein extraction buffers, maximal rate shake
15min is swung, is stood and open after 3min centrifuge tube lid, centrifuge tube is placed in boiling water and heats 10min, taken out centrifuge tube and be placed in 0
In DEG C frozen water, maximal rate concussion 5min after cooling 3min, 4 DEG C of 5000rpm are centrifuged 5min, supernatant are crossed into 0.45 μm of nylon
Filter membrane, the supernatant of filter membrane be transferred in a new 10ml centrifuge tube, add the Tris saturated phenols of 2ml pH 8.0, most
4 DEG C of 12000rpm are centrifuged 20min after big speed concussion 20min, remove supernatant, remove layer phenol and are placed in 50ml centrifuge tubes,
The 0.1M methyl alcohol ammonium acetate solutions of about 5-6 times of volume of phenol layer are added, -20 DEG C are placed after 6h, from -20 DEG C of 4 DEG C of centrifuge tubes of taking-up
15000rpm is centrifuged 20min, and supernatant is carefully removed from, and is precipitated as protein, then albumen precipitation is cleaned into 3 with the acetone of precooling
Secondary, -80 DEG C freeze, and obtain protein dry powder.
2 Separation of Proteins：3 kinds of protein are taken out respectively into 1.5mg protein powders to be placed in 2ml centrifuge tubes, 1ml is added
SDT buffer solution protein powders, take 20 μ l protein solutions in 2ml centrifuge tubes, add 5 μ l5 × sample-loading buffer system
Standby into protein example, be vortexed boiling water bath 10min after mixing, and treats that sample is cooled down, and 3 kinds of soil protein examples take respectively 20 μ l and enter
Row SDS-PAGE electrophoresis, gel specification is 12 × 16 × 18cm, and electrophoretic buffer is added in electrophoresis tank, and concentration glue is poly- for 5%
Acrylamide gel, 80V, electrophoresis 30min, separation gel is 12% polyacrylamide gel, and 120V, electrophoresis 90min, 15mA is permanent
Determining electric current carries out electrophoresis, and room temperature after electrophoresis terminates, unloads film, and coomassie brilliant blue staining, destainer decolourizes.
Fig. 1 is Wang et al., the soil albumen that 2011, Chourey et al., 2010 and the inventive method are extracted
The SDS-PAGE glue figures of matter extracting method.
Fig. 2 is the SDS-PAGE glue figures that this method extracts different soils protein.
As a result show, the protein of the different soils extracted using the present invention, Jing after SDS-PAGE detections, extracted
Preferably, soil protein concentration is high, band is clear for soil protein quality.
Claims (7)
1. a kind of soil method for extracting protein, comprises the steps：
1) soil protein extraction buffer, concussion is added to stand in pedotheque；
2) it is placed in boiling water and heats, cools down in 0 DEG C of frozen water, concussion, centrifugation；
3) by supernatant liquid filtering, Tris saturated phenols, concussion, centrifugation, abandoning supernatant is added to collect in filtered supernatant
Lower floor's phenol solution；
4) methyl alcohol ammonium acetate solution, -20 DEG C of placement 6h-8h are added in lower floor's phenol solution；
5) it is centrifuged, supernatant is removed, collect protein precipitation, then protein precipitation is cleaned with acetone, freeze, obtains albumen
Matter dry powder.
2. method according to claim 1, it is characterised in that：Step 1) in, the soil protein extraction buffer is
Neutral protein extraction buffer, the concretely protein extraction buffer of pH6.8；
The compound method of the protein extraction buffer of the pH6.8 is：Weigh lauryl sodium sulfate 1.25g, trihydroxy methyl
Aminomethane 1.2114g, NaCl 0.8766g, dithiothreitol (DTT) 0.386g, add the dissolving of 70ml distilled waters, are adjusted using concentrated hydrochloric acid
Section pH to pH 6.8, is finally settled to 100ml using distilled water, you can.
3. method according to claim 1 and 2, it is characterised in that：Step 1) in, the pedotheque and soil protein
The proportioning of Extraction buffer is 1.8g-2.2g：5ml；
The speed of the concussion is 3000rpm-3200rpm, and the time is 10-20min；
The time of the standing is 3-5min.
4. the method according to any one of claim 1-3, it is characterised in that：Step 2) in, the time of the heating is
5-15min；
The time of the cooling is 3-5min；
The speed of the concussion is 3000rpm-3200rpm, and the time is 5-10min；
The condition of the centrifugation is：4500-5500rpm centrifugations 75-10min at 4 DEG C.
5. the method according to any one of claim 1-4, it is characterised in that：Step 3) in, described filtration adopts 0.45 μ
The nylon leaching film of m；
The pH value of the Tris saturated phenols is 8.0；
The supernatant is 3-4ml with the proportioning of Tris saturated phenols：2ml；
The speed of the concussion is 3000rpm-3200rpm, and the time is 15-30min；
The condition of the centrifugation is：10000-13000rpm centrifugations 15-20min at 4 DEG C.
6. the method according to any one of claim 1-5, it is characterised in that：Step 4) in, the methyl alcohol ammonium acetate is molten
The concentration of liquid is 0.1M；The methyl alcohol ammonium acetate solution is 5-6 with the volume ratio of phenol solution：1.
7. the method according to any one of claim 1-6, it is characterised in that：Step 5) in, the condition of the centrifugation is：
13000-15000rpm centrifugations 15-20min at 4 DEG C；
The cleaning is carried out repeatedly, concretely 3 times；
The lyophilized temperature is -80 DEG C.
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| CN201710068838.8A CN106632582A (en) | 2017-02-08 | 2017-02-08 | Extraction method of soil protein |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107167353A (en) * | 2017-07-05 | 2017-09-15 | 赛思莱（厦门）生物科技有限公司 | A kind of sample preparation processing method for the grand protein science research of soil |
| CN109810168A (en) * | 2019-03-18 | 2019-05-28 | 龙岩学院 | A kind of efficient soil protein extracting method |
| CN110437301A (en) * | 2019-08-27 | 2019-11-12 | 上海美吉生物医药科技有限公司 | A kind of macro protein extracting method of suitable environmental classes sample and excrement class sample |
-
2017
- 2017-02-08 CN CN201710068838.8A patent/CN106632582A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| SHAONING CHEN等: "Improving soil protein extraction for metaproteome analysis and glomalin-related soil protein detection", 《PROTEOMICS》 * |
| 熊艺: "土壤宏蛋白质组学之土壤蛋白质提取技术的发展", 《土壤》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107167353A (en) * | 2017-07-05 | 2017-09-15 | 赛思莱（厦门）生物科技有限公司 | A kind of sample preparation processing method for the grand protein science research of soil |
| CN107167353B (en) * | 2017-07-05 | 2019-11-26 | 赛思莱(厦门)科技有限公司 | A kind of sample preparation processing method for the macro protein science research of soil |
| CN109810168A (en) * | 2019-03-18 | 2019-05-28 | 龙岩学院 | A kind of efficient soil protein extracting method |
| CN110437301A (en) * | 2019-08-27 | 2019-11-12 | 上海美吉生物医药科技有限公司 | A kind of macro protein extracting method of suitable environmental classes sample and excrement class sample |
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Application publication date: 20170510 |