CN103319566A - Method for extracting fungus total protein - Google Patents
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- CN103319566A CN103319566A CN2013102586145A CN201310258614A CN103319566A CN 103319566 A CN103319566 A CN 103319566A CN 2013102586145 A CN2013102586145 A CN 2013102586145A CN 201310258614 A CN201310258614 A CN 201310258614A CN 103319566 A CN103319566 A CN 103319566A
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Abstract
The invention provides a method for extracting fungus total protein. A simple and feasible method is adopted, the characteristics that the fungal cell wall is thick and tough and the intracellular protein is difficult to release are overcome, and high-quality fungus proteome is successfully extracted. The components of an extracting solution are simple; the extracting solution I comprises 7-8M urea, 2-2.5M thiourea, 1-2m MEDTA (ethylenediamine tetraacetic acid), 10mMTris-HCL (trihydroxy methyl-aminomethane hydrochloride with the pH of 7.4) and 10ul protease inhibitor mixture; the extracting solution II comprises 2-4 percent of CHAPS (3-[3-(cholylaminopropyl) dimethylamino]CHAPS, 0.5 percent of SDS (sodium dodecyl sulfate) and 2-4 percent of DTT (dithiothreitol). The method is simple, convenient and rapid and is low in equipment requirement; and moreover, the extraction process of total protein can be finished within three hours, lots of total protein can be obtained and is used for proteome analysis, and the method has high actual application value.
Description
Technical field
The present invention relates to the extracting method of total protein in the proteomics research field, particularly relate to a kind of efficiently, the extracting method of fungi total protein fast.
Background technology
Fungi (Fungus) belongs to the super guiding principle of fungi in the thallophyte, the microorganism of tool eukaryotic cell type in the biology classification.Along with the mankind to the going deep into of the research of fungi, fungi is closely bound up with the mankind's life.Fungi is extensive in distributed in nature, most favourable to the people, such as wine brewing, sauce processed, fermented feed, microbiotic is made in the farmland getting fat, produces mushroom, food-processing and Chinese herbal medicine source (such as glossy ganoderma, Poria cocos, Cordyceps sinensis etc., all be the product of fungi or itself or utilize the effect of fungi prepared) is provided.But the fungi that couple human disease is also arranged is divided into superficial mycosis and deep fungal, and some fungi autoeciousness can produce toxin and cause the toxic mycosis in grain, feed, food in addition.
Bidirectional electrophoresis technique is that O ' Farrell ' s, Klose and Scheele equal 1975 inventions, and it makes albumen separate because of the difference of iso-electric point first first by isoelectrofocusing; Second by the SDS-polyacrylamide gel electrophoresis, and protein subunit is separated by molecular size range; Separate for twice by iso-electric point and molecular weight, the protein in the mixture separates at two dimensional surface, thereby obtains the information of isoelectric points of proteins and molecular weight.Two dimensional gel electrophoresis is a kind of high-resolution protein stripping technique, is again a kind of collection technique of protein, thereby becomes the core technology of proteomics research.And in the proteome research, can effectively extract target protein from complex sample be the key of carrying out the mass spectroscopy success or not.
People are more and more to the research of fungi now, make the fungi product come into huge numbers of families, for people's life health has been made huge contribution.At present people mostly concentrate on some for the research of fungi and have using value or the mankind are had on the fungi of pathogenic effect, and large multi-method also is on a certain or a certain class bacterial strain, lacks a kind of universal method.Proteomics research for fungi also is in a starting stage at present, therefore is badly in need of a kind of universal method and instructs people to carrying out that the mycoprotein group is studied, thereby make it better serve the mankind.In addition, this method also can be applicable in other experimental implementation of relevant mycoprotein correlative study.
Summary of the invention
The purpose of this invention is to provide the extracting method of fungi total protein in a kind of proteomics research field, the method is efficiently quick.
The extracting method of fungi total protein provided by the invention comprises following step:
(1) cultured thalline is carried out suction filtration and collects, use the PBS(phosphate buffered saline buffer) regather thalline after washing, or adopt the centrifugal thalline of collecting first, then wash thalline with PBS, more centrifugal collection thalline;
(2) an amount of thalline that will collect places the mortar of Liquid nitrogen precooler, fully grinds with liquid nitrogen, thalline is ground to Powdered;
(3) in adding the ratio of grinding rear thalline powder 100-150mg in per 800 μ l extracting solution I, the thalline powder places the extracting solution I after will grinding, fully mixing is placed on and hatches 30-60min on ice, every the 5-7min mixing once, wherein the extracting solution I refers to contain 7 ~ 8M urea, 2 ~ 2.5M thiocarbamide, 1 ~ 2mM EDTA(ethylenediamine tetraacetic acid (EDTA) between incubation period), 10mM Tris-HCL(Tri(Hydroxymethyl) Amino Methane Hydrochloride, pH7.4), the solution of 10ul protease inhibitor cocktail (100X); In order to guarantee that albumen is not degraded, need during the weighing thalline first extracting solution to be placed centrifuge tube, want rapid mixing behind the adding thalline powder, place on ice again, and mixing is put upside down once in the interval;
(4) add in the solution of ratio after step (3) is hatched of extracting solution II of 200 μ l in per 800 μ l extracting solution I and add the extracting solution II, putting upside down mixing is placed on and hatches 20-30min on ice, every the 3-5min mixing once, wherein the extracting solution II is for containing 2 ~ 4%CHAPS(3-[3-(courage acyl aminopropyl) dimethylamino between incubation period] the propanesulfonic acid inner salt), 0.5%2 ~ 4%SDS (sodium lauryl sulphate), 2 ~ 4%DTT(dithiothreitol (DTT)) mixing solutions;
(5) will extract rear liquid places centrifuge tube in low-temperature and high-speed refrigerator centrifugal treating;
(6) get supernatant and abandon precipitation, supernatant and the total protein for extracting;
(7) with the total protein packing of extracting, place refrigerator and cooled to freeze to preserve or carry out next step experiment.
The PBS that uses in the step in the inventive method (1) refers to 1 * PBS of precooling, and pH is 7.4, and washing times is 2-3 time.
Centrifugal collection thalline refers at 4 ℃, the centrifugal 10-15min of 6000-10000rmp in the inventive method.
Adopt 4 ℃, the centrifugal 15min of 13000rmp when centrifugal in the step in the inventive method (5), the supernatant after centrifugal carefully changes in the new centrifuge tube with pipettor, if but be drawn onto the precipitation recentrifuge and remove precipitation.
In the step in the inventive method (8) albumen that extracts is packed as some parts, can does once experiment with every part and be advisable, avoid multigelation to the infringement of albumen.
Above-mentioned percentage concentration is mass volume ratio concentration (w/v, g/ml), and centrifugal speed represents with rotating speed.
The inventive method is fit to extract total protein from fungi, that filamentous fungus or yeast all have good effect, the method is simple and efficient, just can extract a large amount of fungi total proteins with conventional laboratory equipment and reagent, has larger actual application value.
Description of drawings
Fig. 1 is the SDS-PAGE detection schematic diagram that Lung biopsy extracts the Mortierella isabellina total protein, and among the figure: swimming lane 1 is the total protein that method 1 is extracted; Swimming lane 2 is the total protein that method 2 is extracted; Swimming lane 3 is the total protein that method 3 is extracted; Swimming lane 4 is the total protein that method 4 is extracted; Swimming lane 5 is the total protein that method 5 is extracted; M is albumen Marker;
Fig. 2 is the contrast collection of illustrative plates of method 2 and the inventive method, among the figure: the 1st, the total protein that method 2 is extracted; The 2nd, the total protein that the inventive method is extracted; M is albumen Marker;
Fig. 3 is for to extract the total protein collection of illustrative plates of six all fungies with method of the present invention, among the figure: M is albumen Marker, and 1 is that the total protein, 2 of Mortierella isabellina is the total protein of aspergillus niger for the total protein of mould, 8 for the mould total protein of wood, 5 for the total protein of Fusarium oxysporum, 4 for the total protein of yeast saccharomyces cerevisiae, 3;
Fig. 4 is the Two-dimensional Gel Electrophoresis of the Mortierella isabellina albumen of the inventive method extraction.
Embodiment
Below by drawings and Examples the present invention is described in further detail, but protection domain of the present invention is not limited to described content, reagent is commercially available conventional reagent or the reagent that makes according to a conventional method if no special instructions among the embodiment.
The reagent that uses in the present embodiment is as follows:
Protease inhibitor cocktail solution (100X) is purchased from Shanghai and gives birth to worker biotech firm, molecular weight of albumen standard (Thermo Fisher), dithiothreitol (DTT) DTT(Amresco), urea (Amersham), thiocarbamide (Amersham), CHAPS (Usb), Tris-base(Amresco), the HCl(Guangzhou Chemical Reagent Factory), acrylamide (Amresco), methylene diacrylamide (Sigma), EDTA(Sigma), immobilized ph gradient strip drystrip(24cm, pH4-7) (GE), all the other are domestic analytical reagent; The two-dimensional electrophoresis solution such as two-dimensional electrophoresis urea hydrating fluid, SDS balance liquid, agarose sealing liquid are all prepared with reference to GE company " two-dimensional electrophoresis principle and method "; 1 * PBS damping fluid is prepared by following concentration: KCl 0.2g/L, NaCl 8.0g/ L, KH
2PO
40.27g/L, Na
2PO
41.42g/L, pH7.4.
Embodiment 1: Lung biopsy and the inventive method are extracted the unidirectional comparative experiments of Mortierella isabellina total protein effect
Through searching data, select the total protein that Lung biopsy extracts Mortierella isabellina, come the quality of comparison extracting method effect with SDS-PAGE, concrete method may further comprise the steps:
1, the cultivation of thalline
The spore of Mortierella isabellina is in the PDA liquid nutrient medium on the picking inclined-plane, cultivates 72h under 28 ℃, 150rmp condition;
2, Lung biopsy extracts the total protein of Mortierella isabellina, and Lung biopsy is as described below:
Method 1: cultured thalline is collected thalline with suction filtration, with ultrapure water washing 3 times, refilter collection, a part of thalline of collecting fully grinds in liquid nitrogen, the mycelia powder of getting after 100mg grinds is put into 1ml lysate (20 mM Tris-HCl pH 7.4,150 mM NaCl, 1%(w/v, g/ml) NP-40,0.5%(w/v, g/ml) Sodium desoxycholate, 0.1%(w/v, g/ml) SDS, 1 * protease inhibitor cocktail) in, mix rapidly fully, hatch 1h on ice, during put upside down mixing every 5min; Above-mentioned centrifuge tube is put into centrifuge tube centrifugal (4 ℃, 24000g, 30min), supernatant is changed in the centrifuge tube of new precooling and carry out follow-up experiment.
Method 2: centrifugal collection thalline, ultrapure water washing (3000r/min, centrifugal 19min) 3 times; Take by weighing thalline with the 2ml centrifuge tube, add 100ul extracting solution I (7.5M urea, 2.5M thiocarbamide, 1.5mM EDTA, 10mM Tris, 20mM PMSF, 2mg/L Dnase, 5mg/L Rnase) according to every 200mg thalline; Room temperature effect 15-20min; In aforesaid liquid, add 300mg pickling glass pearl (diameter 0.5mm), in centrifuge tube, fully grind approximately 10min with glass stick after, vortex concussion 10min(1800r/min on the vortex vibrator); Add extracting solution II (2% CHAPS, 1%DTT) 25ul, vortex 30s * 6 time, during interval 1min place on ice; The centrifugal 10min of 10000r/min abandons precipitation and gets supernatant.
Method 3: cultured thalline suction filtration is collected, with ultrapure water washing 2-3 time; Place liquid nitrogen fully to grind a part of thalline of collecting; 1.5ml centrifuge tube with precooling takes by weighing 100mg mycelia powder, adds the resuspended thalline of 1ml lysate (50mM Tris-Hcl, 1mM EDTA, 50mM NaCl, 0.1% Triston X-100,1mM DTT, 1mM PMSF, pH8.0); 4 ℃ of ice bath 30min, during every the 5min mixing once; Utilize ultrasonic cell disruption instrument broken thalline (750W, ultrasonic 6s, interval 1s, ultrasonic 20 times) in ice bath; With the centrifugal 20min of high speed freezing centrifuge (12000r/min), abandon precipitation and get supernatant.
Method 4: the thalline ultrafiltration of collecting is collected, with ultrapure water washing 3 times; Place liquid nitrogen to grind 3-5 time a part of thalline of collecting, make it become fine powder; Add helicase and the 1mol/L DTT 50ul of 300mg, add again 25mmol/L potassium phosphate buffer (ph7.9, PBS) to 20ml, vortex mixing, 37 ℃ of water-bath 3h.4 ℃ centrifugal (9300g, 5min), supernatant discarded repeats 2 times, cleans helicase; Add lysate (8M urea, 200mM yellow soda ash) 400ul in precipitation, the piping and druming mixing is hatched 30min on ice, concuss 20min; Sample blending liquid is got supernatant 300ul in-70 ℃ of preservations in 4 ℃ centrifugal (15700g, 20min).
Method 5: the thalline ultrafiltration of collecting is collected, with ultrapure water washing 2-3 time; Getting the 1g thalline grinds in liquid nitrogen 5 times; Get powder in the 1.5ml centrifuge tube, add the 10%TCA-acetone soln (containing 20mM DTT) of precooling ,-20 ℃ of placements are spent the night with protein precipitation behind the mixing; In 4 ℃ of centrifugal (13400g) 25min, abandon supernatant, add again acetone soln (containing 20mM DTT) the 1ml washing of precooling in the precipitation, 4 ℃ of centrifugal (13400g) 25min abandon supernatant, repeat 2 times; Settling chamber's warm air is done, treated that residual acetone waves to the greatest extent, precipitation is dried to fine powder.In precipitation, add the dielectrophoresis sample lysate 400ul contain urea, thiocarbamide, room temperature lixiviate 2h, during repeatedly use aseptic rifle head pressure-vaccum, solution is fully contacted with precipitating; 4 ℃ of centrifugal (13400g) 20min of sample solution.Get supernatant (approximately 300ul) in-70 ℃ of preservations.
The total protein of the Mortierella isabellina that 3, method 1-5 is extracted carries out the unidirectional electrophoresis detection of SDS-PAGE
The total protein that method 1-5 in the step 2 is extracted carries out the SDS-PAGE detection according to a conventional method, the result as shown in Figure 1, as can be seen from the figure the protein concn that extracts of method 4 and method 5 is larger, but there is degraded in some protein that method 4 is extracted, protein band is unintelligible, method 5 is poor for some low-abundance proteins extraction effects, and the large protein band of molecular weight is few, although the protein concn that method 2 is extracted is relatively low, but its protein band that extracts is many, be evenly distributed, can also extract some low-abundance protein.
Table 1: the total protein concentration result that Lung biopsy extracts
4, adopt method of the present invention to carry out the extraction of Mortierella isabellina total protein, comprise following step:
1) cultured thalline being carried out suction filtration and collect, is 1 * PBS washing 3 times of 7.4 precooling, again suction filtration collection thalline with pH;
2) thalline of collecting is placed mortar with Liquid nitrogen precooler, fully grind 5 times with liquid nitrogen, thalline is ground to tiny Powdered;
3) thalline after getting 100mg and grinding places the centrifuge tube of the 1.5ml of precooling, add 800ul extracting solution I (7.5M urea, 2.5M thiocarbamide, 1.5mM EDTA(ethylenediamine tetraacetic acid (EDTA)), 10mM Tris-HCL(Tri(Hydroxymethyl) Amino Methane Hydrochloride, pH7.4), the 10ul protease inhibitor cocktail, fully mixing is placed on and hatches 60min on ice, during put upside down mixing once every 5min;
4) after step 3) is hatched solution in add 200ul extracting solution II (2%(w/v, g/ml) CHAPS(3-[3-(courage acyl aminopropyl) dimethylamino] the propanesulfonic acid inner salt), 0.5%(w/v, g/ml) SDS (sodium lauryl sulphate), 2%(w/v, g/ml) DTT(dithiothreitol (DTT))), put upside down mixing and be placed on and hatch 30min on ice, during put upside down mixing once every 5min;
5) will extract after liquid place centrifuge tube in low-temperature and high-speed refrigerator centrifugal treating, adopt 13000rmp, 4 ℃ of centrifugal 15min when centrifugal, the supernatant after centrifugal carefully changes in the new centrifuge tube with pipettor;
6) get supernatant and abandon precipitation, supernatant and the total protein for extracting;
7) with the total protein packing of extracting, place refrigerator and cooled to freeze to preserve or carry out next step experiment.
5, the total protein that the inventive method is extracted carries out the unidirectional electrophoresis detection of SDS-PAGE
Can find out that from electrophoretogram (Fig. 2) total protein concentration that present method is extracted is significantly improved, low-abundance protein band concentration obviously increases, and the concentration height do not cause protein degradation, to the destruction less of albumen, extracts quality and significantly improves.
Table 2: Lung biopsy and the inventive method are extracted the comparison of protein concentration
Embodiment 2: adopt the inventive method that Mortierella isabellina is carried out the experiment that total protein extracts under the different extraction conditions
(1) extracting method is with embodiment 1, and difference is: the extracting solution I is 7.5M urea, 2.5M thiocarbamide, 1.5mM EDTA, 10mM Tris-HCL(pH7.4), the 10ul protease inhibitor cocktail, incubation time is 50min, every the 6min mixing once; The extracting solution II is 2%CHAPS, 0.5%SDS, 2%DTT, hatches 25min, between incubation period every the 4min mixing once;
(2) extracting method is with embodiment 1, difference is: the extracting solution I is 7M urea, 2M thiocarbamide, 1mM EDTA(ethylenediamine tetraacetic acid (EDTA)), 10mM Tris-HCL(Tri(Hydroxymethyl) Amino Methane Hydrochloride, pH7.4), the 10ul protease inhibitor cocktail, incubation time is 40min, every the 7min mixing once; The extracting solution II is 2%CHAPS(3-[3-(courage acyl aminopropyl) dimethylamino] the propanesulfonic acid inner salt), 0.5%SDS (sodium lauryl sulphate), 2%DTT(dithiothreitol (DTT)), hatch 20min, between incubation period every the 3min mixing once;
(3) extracting method is with embodiment 1, difference is: the extracting solution I is 8M urea, 2.5M thiocarbamide, 2mM EDTA(ethylenediamine tetraacetic acid (EDTA)), 10mM Tris-HCL(Tri(Hydroxymethyl) Amino Methane Hydrochloride, pH7.4), the 10ul protease inhibitor cocktail, incubation time is 30min, every the 5min mixing once; The extracting solution II is 4%CHAPS(3-[3-(courage acyl aminopropyl) dimethylamino] the propanesulfonic acid inner salt), 0.5%SDS (sodium lauryl sulphate), 4%DTT(dithiothreitol (DTT));
(4) extracting method is with embodiment 1, difference is: 4 ℃ first, the centrifugal 12min of 7000rmp collects thalline, then use 1 * PBS washing thalline 2 times, centrifugal collection thalline again, the extracting solution I is 7.5M urea, 2 M thiocarbamides, 1.5mM the EDTA(ethylenediamine tetraacetic acid (EDTA)), 10mM Tris-HCL(Tri(Hydroxymethyl) Amino Methane Hydrochloride, pH7.4), 10ul protease inhibitor cocktail, extracting solution II are 3%CHAPS(3-[3-(courage acyl aminopropyl) dimethylamino] the propanesulfonic acid inner salt), 0.5%SDS (sodium lauryl sulphate), the 3%DTT(dithiothreitol (DTT));
(5) extracting method is with embodiment 1, difference is: 4 ℃ first, the centrifugal 10min of 10000rmp collects thalline, then use 1 * PBS washing thalline 2 times, centrifugal collection thalline again, the extracting solution I is 7M urea, 2.5M thiocarbamide, 1.5mM the EDTA(ethylenediamine tetraacetic acid (EDTA)), 10mM Tris-HCL(Tri(Hydroxymethyl) Amino Methane Hydrochloride, pH7.4), 10ul protease inhibitor cocktail, extracting solution II are 4%CHAPS(3-[3-(courage acyl aminopropyl) dimethylamino] the propanesulfonic acid inner salt), 0.5%SDS (sodium lauryl sulphate), the 3%DTT(dithiothreitol (DTT)).
Table 3: the comparison of extracting total protein concentration under the different condition
As can be seen from the table, in the inventive method effect good to having of Mortierella isabellina total protein under different condition.
Embodiment 3: different thalline powder additions are to the comparison of extraction effect
The thalline powder that adds respectively 100mg, 110mg, 120mg, 130mg, 130mg, 150mg by method of the present invention among the embodiment 1 carries out the comparison of total protein concentration, the results are shown in Table 4.
Table 4: different thalline powder additions are to the comparison of extraction effect
As can be seen from the table, thalline powder addition can not obviously improve total protein concentration.
Embodiment 4: adopt method of the present invention to extract the total protein of six kinds of fungies
The method of current series invention is extracted the total protein of six kinds of fungies, method is with extracting method among the embodiment 1, bacterial classification comprises that common Mortierella isabellina, yeast saccharomyces cerevisiae, Fusarium oxysporum, wood are mould, mould and aspergillus niger, then the effect of its extraction is carried out the unidirectional detection of SDS-PAGE, detected result as shown in Figure 3, as can be seen from the figure, method of the present invention has good extraction effect to these six kinds of fungies, illustrates that present method can be applicable to filamentous fungus and unicellular yeast.The total protein concentration that extracts six kinds of fungies sees Table 5.
Table 5: six kinds of fungi total protein contents that extract with method of the present invention compare
Embodiment 5: select the Mortierella isabellina total protein that extracts to carry out two-dimensional electrophoresis and detect
Pre-treatment: the total protein that extracts is carried out dialysis treatment, to remove salt ion to the impact of two-dimensional electrophoresis, employing molecular retention amount is 3500 dialysis tubing during dialysis, dialysis buffer liquid is the Tris damping fluid of 50mM, pH7.4, change a dialyzate every 2h front twice, dialyzed overnight for the third time, its whole process places 4 ℃ to carry out; The concentration of total protein reduces after the dialysis, but detects through SDS-PAGE, but can find out from collection of illustrative plates, and the band of Before and after dialysis protein is consistent, does not have considerable change, illustrates that dialysis treatment is less on the impact of protein, can adopt dialysis treatment.
Operation steps: the total protein of getting after the 120mg dialysis adds hydrating fluid to 450ul, with pipettor all with the glue groove that places the aquation dish in, glue (24cm, pH4-7) faced down places in the glue groove room temperature aquation 16h; Aquation is transferred to adhesive tape in the focusing dish after finishing, adopt Ettan IPGphor III to carry out first to isoelectrofocusing, the condition that focuses on is: S1 stp 300V 0:30Hr, S2 stp 700V 0:30Hr, S3 stp 1500V 1:30Hr, S4 grd 9000V 3:00Hr, S5 stp 9000V 5:00Hr, the used total voltage of whole during focusing: 64KVh; The upper limit current of adhesive tape is the 50uA/ adhesive tape, and focus temperature is 20 ℃.Focus on and finish to be placed on Balance Treatment in the equilibration tube that the SDS level pad is housed, balance twice, each 15min adds 1% DTT the first time, adds 2.5% IAA for the second time; Balance finishes to be placed on to infiltrate in the electrophoretic buffer, then place on the gel, agarose sealing liquid sealing with 0.5%, be transferred to carry out in the ETTAN DALTsix system second to electrophoresis, the control temperature is 15 ℃, its condition is S1 2W/gel, 45min, and S2 17W/gel is until tetrabromophenol sulfonphthalein is gone to the end; Electrophoresis places the dyeing dish to carry out cma staining gel after finishing; Gel after the dyeing is scanned, first scanner is wiped clean before the scanning, at some water of spraying on glass, gel is transferred on the scanner again, guarantee that the direction of every glue scanning is identical, scan pattern is the scanning of 256 GTGs perspective, and resolving power is 200dpi.
Fig. 4 is the Two-dimensional Gel Electrophoresis of Mortierella isabellina total protein, can find out from the 2D electrophorogram, the total protein that the inventive method is extracted is through after the dialysis treatment, be suitable for the analysis of 2D electrophoresis, the protein spots that obtains is relatively many, through software detection 1983 protein spots is arranged approximately, is fit to carry out the research of proteomics, therefore present method can be applicable to the Two-dimensional Electrophoresis Analysis of other fungi, has a extensive future.Method of the present invention adopts traditional liquid nitrogen grinding wall-breaking method, and not only cost is low but also easy and simple to handle; The adding of urea and thiocarbamide can interrupt in the protein molecule and intermolecular various chemical bonds, polypeptide is stretched, increased the solubleness that non-polar molecule comprises amino acid side chain, having reduced the hydrophobic interaction thiocarbamide can assist urea to the property of protein, can increase simultaneously the solubleness of membranin, make the preparation sample comprise more complete protein group; DTT can protected protein matter sulfydryl is not oxidized freely, thereby avoid focusing and the sex change of protein, urea can assist DTT to carry out the reduction of disulfide linkage simultaneously; CHAPA can the solubilising membranin, and the native state of protected protein matter is removed the interaction between the protein-protein; SDS is an important solvent in the protein extraction process as the denaturing agent solubility promoter, can the saboteur in and intermolecular hydrogen bond destroying two, three grades of results of protein, thereby increase the solubleness of protein; Protease inhibitor cocktail can suppress the activity of multiple protein enzyme, can well keep the integrity of albumen, and effect is better than using separately PMSF.Simple and convenient extraction of the present invention is quick, adopt cheap reagent and not high to equipment requirements commonly used, the advantage such as simple to operate except having, that cost is low, save time, and can be used for multiple fungi, wide spectrum extracting method for a kind of fungi total protein, have larger actual application value, will promote the development of Fungal Protein group.
Claims (5)
1. the extracting method of a fungi total protein may further comprise the steps:
(1) cultured mycothallus is carried out suction filtration and collect, regather thalline after washing with the PBS damping fluid, or the centrifugal collection thalline of elder generation, then wash thalline with PBS, more centrifugal collection thalline;
(2) thalline of collecting is placed the mortar of Liquid nitrogen precooler, fully grind with liquid nitrogen, thalline is ground to Powdered;
(3) in adding the ratio of grinding rear thalline powder 100-150mg in per 800 μ l extracting solution I, the thalline powder places the extracting solution I after will grinding, fully mixing is placed on and hatches 30-60min on ice, every the 5-7min mixing once, wherein the extracting solution I refers to contain the solution of 7 ~ 8M urea, 2 ~ 2.5M thiocarbamide, 1 ~ 2mM ethylenediamine tetraacetic acid (EDTA), 10mM Tris-HCL, 10ul protease inhibitor cocktail between incubation period;
(4) add in the solution of ratio after step (3) is hatched of extracting solution II of 200 μ l in per 800 μ l extracting solution I and add the extracting solution II, put upside down to place again behind the mixing and hatch 20-30min on ice, every the 3-5min mixing once, wherein the extracting solution II is for containing 2 ~ 4% 3-[3-(courage acyl aminopropyl) dimethylamino between incubation period] solution of propanesulfonic acid inner salt, 0.5% sodium lauryl sulphate, 2 ~ 4% dithiothreitol (DTT);
(5) place centrifuge tube in low-temperature and high-speed refrigerator centrifugal treating the liquid after step (4) processing;
(6) get supernatant and abandon precipitation, supernatant is the fungi total protein that extracts;
(7) with the total protein packing of extracting, place refrigerator and cooled to freeze to preserve or carry out next step experiment.
2. the extracting method of described fungi total protein according to claim 1, it is characterized in that: the PBS that uses in the step (1) refers to 1 * PBS of precooling, and pH is 7.4, and washing times is 2-3 time.
3. the extracting method of described fungi total protein according to claim 1, it is characterized in that: centrifugal collection thalline refers at 4 ℃, the centrifugal 10-15min of 6000-10000rmp.
4. the extracting method of described fungi total protein according to claim 1 is characterized in that: centrifugal treating is centrifugal 15min under 4 ℃, the condition of 13000rmp in the step (5).
5. the extracting method of described fungi total protein according to claim 1-5, it is characterized in that: fungi is filamentous fungus or yeast.
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