CN111718394A - Method for extracting sugarcane tissue denatured protein based on BPP method - Google Patents
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- 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
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Abstract
The invention discloses a method for extracting sugarcane tissue denatured protein based on a BPP method, which belongs to the technical field of denatured protein extraction. The invention has broad spectrum, short single time of vortex oscillation after using ultrasound, can effectively avoid the fracture of protein structure and introduce new structure, effectively solves the problem of salt ions in the preparation of two-dimensional electrophoresis protein samples, can be widely applied to various plants and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of denatured protein extraction, in particular to a method for extracting sugarcane tissue denatured protein based on a BPP method.
Background
In proteomics research, the most effective method at present is to separate proteins by a two-dimensional electrophoresis technique (2-DE), and then perform mass spectrometry on the separated proteins. Since the protein extraction methods for dielectrophoresis are very different depending on the plant species and tissue site, the extraction of high quality proteins is one of the most challenging tasks in dielectrophoresis and later mass spectrometric identification. Plant tissues, particularly sugarcane tissues, usually contain a large amount of recalcitrant substances such as polysaccharide, quinones, polyphenol and the like, so that the effect of protein separation by two-dimensional electrophoresis is influenced, and further, the later-stage mass spectrum identification work is interfered.
The TCA method is a classical and effective protein sample preparation method suitable for two-dimensional electrophoresis, and has the greatest advantage of rapidly and effectively inhibiting the activity of various proteases. But the method is mainly applied to animals and certain model plants; in addition, the obvious drawbacks of this method are that proteins precipitated via TCA are difficult to dissolve and there is no way to remove salt ions from the sample.
Disclosure of Invention
The invention aims to provide a method for extracting sugarcane tissue denatured protein based on a BPP method, which solves the technical problems mentioned in the background technology.
A preparation method of a sugarcane tissue denatured protein extraction method based on a BPP method comprises the following steps:
step 1: preparing a test instrument and a fresh tissue sample;
step 2: grinding the tissue sample in the environment of liquid nitrogen;
and step 3: mixing and centrifuging the ground tissue sample by using a BPP (BPP) extracting solution to obtain a mixed solution;
and 4, step 4: and washing the mixed solution to extract the protein.
Further, the specific process of step 1 is as follows:
step 1.1: preparing liquid nitrogen, pouring the liquid nitrogen from a liquid nitrogen tank to a vacuum cup, preparing a mortar and a pestle, pouring anhydrous alcohol covering the surface, and firing for later use;
step 1.2: prepare fresh tissue samples or remove tissue samples from a cryo-refrigerator, leave in ice-boxes, weigh immediately with the container containing the sample, and record the weight.
Further, the specific process of step 2 is as follows:
step 2.1: before grinding, precooling a mortar and a pestle by liquid nitrogen in advance; preparing a centrifugal tube, marking the name of a sample, and putting the centrifugal tube into a vacuum cup filled with liquid nitrogen for precooling;
step 2.2: taking a weighed tissue sample, putting the tissue sample into a precooled mortar, quickly stirring and grinding the tissue sample into a dry powder, and replenishing liquid nitrogen while grinding to keep the sample at a low temperature in the grinding process;
step 2.3: after grinding, adding liquid nitrogen just submerging the tissue sample, precooling a stainless steel medicine spoon in the liquid nitrogen, collecting the powder splashed in the mortar to the bottom of the mortar by using the medicine spoon, collecting the tissue powder into the corresponding precooled centrifugal tube, and putting the tissue powder into the liquid nitrogen again for later use;
step 2.4: empty tinfoil and centrifuge tubes were weighed to 0.1g, and sample powder was added to the tinfoil tubes and weighed, and the tissue sample mass calculated.
Further, the specific process of step 3 is as follows:
step 3.1: calculating the dosage of the BPP extracting solution according to the mass of the tissue sample, wherein the dosage of the BPP extracting solution is 7ml of BPP extracting buffer solution used for each gram of the tissue sample. The BPP extraction buffer comprises 100mmol/L EDTA,50mmol/L borax, 50mmol/L vitamin C, 1% PVPP, 1% Triton X-100, 0.1% beta-mercaptoethanol, 25% sucrose, 10mmol/L tris base, Ph7.8).
Step 3.2: adding the ground tissue-divided powder into a BPP buffer according to the calculation mode of 3ml of BPP buffer/1g of tissue sample, pouring the mixture into the solution, turning upside down and uniformly mixing the mixture, and putting the mixture into a refrigerator for hydration at 4 ℃ for 1h for later use; BPP buffer is BPP extraction buffer.
Step 3.3: adding dithiothreitol to a concentration of 5mM before use; extracting for the first time, adding phenylmethylsulfonyl fluoride with the concentration of 1mM before use, mixing uniformly, adding into the tissue powder, mixing uniformly, and performing vortex oscillation for 5min after ultrasonic treatment;
step 3.4: adding equal volume of Tris saturated phenol, wherein the pH value of the Tris saturated phenol is more than 7.8, and vortex mixing for 10min at room temperature;
step 3.5: centrifuging with a centrifuge at 4 deg.C and 12500rpm for 15 min;
step 3.6: preparing a new centrifuge tube, centrifuging, adding phenol, adding equal-volume BPP Buffer, adding dithiothreitol until the final concentration is 5mM before the BPPBuffer is used, mixing uniformly, and vortex mixing at room temperature for 10 min;
step 3.7: centrifuging with a centrifuge at 4 deg.C and 12500rpm for 15 min;
step 3.8: prepare a new centrifuge tube, centrifuge, add phenol, add 5 volumes of pre-cooled saturated ammonium sulfate solution in methanol, precipitate overnight at-20 ℃.
Further, in the step 3.3, the number of the ultrasonic post-vortex oscillations is 30, the time of each post-vortex oscillation is 10s, and the gap time is 10 s.
Further, the ammonium sulfate methanol solution in step 3.8 is prepared by mixing 6.605g ammonium persulfate powder and 500ml methanol.
Further, the specific process of step 4 is as follows:
step 4.1: subpackaging the mixed solution on ice, and performing centrifugal precipitation by using a centrifugal machine at 4 ℃ and 12500rpm for 15 min;
step 4.2: discarding the supernatant, adding methanol pre-cooled at-20 deg.C into each tube, scattering, resuspending, precipitating, balancing, centrifuging at 4 deg.C for 15min at 12500rpm by use of a centrifuge;
step 4.3: repeating the step 4.2 once, discarding the supernatant, and airing the precipitate;
step 4.4: adding lysate into the precipitate, controlling the concentration of the protein solution at 5-8ug/ul, performing vortex oscillation for 30 times after ultrasonic treatment, wherein the vortex oscillation time is 10s after each time, and the gap time is 10 s;
step 4.5: and (3) centrifuging by using a centrifuge at the temperature of 4 ℃ and the centrifugal speed of 12500rpm for 15min, and collecting supernatant to obtain the denatured whole protein lysate.
Further, the air-drying precipitation in the step 4.3 is air-drying, and the air-drying degree is based on the fact that the protein blocks begin to have 1-3mm cracks.
By adopting the technical scheme, the invention has the following technical effects:
the invention has broad spectrum, short single time of vortex oscillation after using ultrasound, can effectively avoid the fracture of protein structure and introduce new structure, effectively solves the problem of salt ions in the preparation of two-dimensional electrophoresis protein samples, can be widely applied to various plants and has wide application prospect.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments are given and the present invention is described in further detail. It should be noted, however, that the numerous details set forth in the description are merely for the purpose of providing the reader with a thorough understanding of one or more aspects of the present invention, which may be practiced without these specific details.
A preparation method of a sugarcane tissue denatured protein extraction method based on a BPP method comprises the following steps:
1.1 preparation work
1.1.1 liquid nitrogen preparation, pouring liquid nitrogen from a liquid nitrogen tank to a vacuum cup, preparing a mortar and a pestle, pouring absolute ethyl alcohol covering the surface, and burning for later use.
1.1.2 preparation of fresh tissue samples were taken from a cryogenic refrigerator, kept in ice box, weighed immediately with the container containing the sample, and the weight was recorded.
Note that: immediately after the sample was removed, the protein extraction operation was started, minimizing the residence time in the ice box.
1.2 liquid nitrogen milling
1.2.1 before the grinding begins, precooling a mortar and a pestle by liquid nitrogen in advance; a50 ml centrifuge tube is prepared, the sample name is marked, and the centrifuge tube is placed into a vacuum cup filled with liquid nitrogen for precooling.
1.2.2 taking the weighed tissue sample, putting the tissue sample into a precooled mortar, quickly stirring and grinding the tissue sample into dry powder, and supplementing liquid nitrogen in time during grinding to keep the sample at low temperature.
1.2.3 after grinding is complete, add the appropriate amount of liquid nitrogen (just past the tissue sample), mixIs not limited toPrecooling the stainless steel medicine spoon in liquid nitrogen, concentrating the powder splashed in the mortar to the bottom of the mortar by using the medicine spoon, quickly collecting the tissue powder into the corresponding precooled 50ml centrifugal tube, and putting the centrifugal tube into the liquid nitrogen again for later use.
1.2.4 weigh the empty containers (tinfoil and centrifuge tubes, etc.) to the nearest 0.1g and calculate the tissue sample mass.
1.3BPP extraction of proteins
1.3.1.1 calculating the dosage of BPP extract according to the tissue sample mass, and calculating according to the tissue standard of 1g BPP extract of 7ml/g (total volume of the first extraction and the second extraction, total buffer amount of the two extractions).
1.3.1.2 grinding, adding the tissue-divided powder into BPPbuffer according to the calculation mode of 3ml BPP buffer/1g tissue sample, pouring into the solution, turning upside down, fully mixing, and putting into a refrigerator for hydration at 4 ℃ for 1h for standby. Immediately before use, DTT is added to a final concentration of 5 mM; extracting for the first time (before adding saturated phenol), adding PMSF (PMSF) with final concentration of 1mM before use, mixing, adding into tissue powder, mixing, performing ultrasonic treatment (ultrasonic treatment for 10s, interval 10s, 30 times), and performing vortex oscillation for 5min after ultrasonic treatment.
1.3.1.3 Add an equal volume (no BPP buffer volume consistent) of Tris saturated phenol (pH >7.8) and vortex mix for 10min at room temperature.
Centrifuge at 12500rpm for 15min at 1.3.1.44 ℃.
1.3.1.5 prepare a new 50ml centrifuge tube, after centrifugation, transfer phenol phase into it, add equal volume of BPPBuffer (not the first BPP buffer volume is consistent, just before use, add DTT, final concentration is 5mM), mix well, then vortex at room temperature for 10 min.
1.3.1.64 ℃, 12500rpm,15 min.
1.3.1.7 preparing a new 50ml centrifuge tube, transferring phenol phase into the centrifuge tube after centrifugation (calculating volume when taking ), adding 5 times volume of precooled supersaturated ammonium sulfate methanol solution (6.605g ammonium persulfate powder +500ml methanol), and precipitating at 20 ℃ overnight;
1.3.2 protein washes
1.3.2.1 taking protein (white precipitate can be seen), and subpackaging on ice; the pellet was centrifuged at 12500rpm for 15min at 4 ℃.
1.3.2.2 discarding the supernatant, adding methanol pre-cooled at-20 ℃ into each tube to break up the heavy suspension precipitate, and balancing at 4 ℃ and 12500rpm for 15 min; this step is repeated.
1.3.2.3 discarding the supernatant, adding acetone pre-cooled at-20 deg.C into each tube to break up the heavy suspension precipitate, balancing, and standing at 4 deg.C and 12500rpm for 15min
1.3.2.4 discard the supernatant and air dry the precipitate. (Note: air drying is good at the onset of small cracks in the protein cake).
1.3.2.5 adding a proper amount of lysate according to the precipitation volume, and controlling the concentration of the protein solution to be 5-8ug/ul as much as possible, and carrying out ultrasonic treatment (ultrasonic treatment for 10s, interval 10s and ultrasonic treatment for 30 times). Collecting protein solution, centrifuging at 4 deg.C and 12500rpm for 10min, and collecting supernatant to obtain denatured whole protein lysate.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (8)
1. A method for extracting sugarcane tissue denatured protein based on a BPP method is characterized by comprising the following steps: the method comprises the following steps:
step 1: preparing a test instrument and a fresh tissue sample;
step 2: grinding the tissue sample in the environment of liquid nitrogen;
and step 3: mixing and centrifuging the ground tissue sample by using a BPP (BPP) extracting solution to obtain a mixed solution;
and 4, step 4: and washing the mixed solution to extract the protein.
2. The method for extracting the sugarcane tissue denatured protein based on the BPP method according to claim 1, which is characterized in that: the specific process of the step 1 is as follows:
step 1.1: preparing liquid nitrogen, pouring the liquid nitrogen from a liquid nitrogen tank to a vacuum cup, preparing a mortar and a pestle, pouring anhydrous alcohol covering the surface, and firing for later use;
step 1.2: prepare fresh tissue samples or remove tissue samples from a cryo-refrigerator, leave in ice-boxes, weigh immediately with the container containing the sample, and record the weight.
3. The BPP method-based preparation method of sugarcane tissue denatured protein extraction method according to claim 1, characterized in that: the specific process of the step 2 is as follows:
step 2.1: before grinding, precooling a mortar and a pestle by liquid nitrogen in advance; preparing a centrifugal tube, marking the name of a sample, and putting the centrifugal tube into a vacuum cup filled with liquid nitrogen for precooling;
step 2.2: taking a weighed tissue sample, putting the tissue sample into a precooled mortar, quickly stirring and grinding the tissue sample into a dry powder, and replenishing liquid nitrogen while grinding to keep the sample at a low temperature in the grinding process;
step 2.3: after grinding, adding liquid nitrogen just submerging the tissue sample, precooling a stainless steel medicine spoon in the liquid nitrogen, collecting the powder splashed in the mortar to the bottom of the mortar by using the medicine spoon, collecting the tissue powder into the corresponding precooled centrifugal tube, and putting the tissue powder into the liquid nitrogen again for later use;
step 2.4: empty tinfoil and centrifuge tubes were weighed to 0.1g, and sample powder was added to the tinfoil tubes and weighed, and the tissue sample mass calculated.
4. The BPP method-based preparation method of sugarcane tissue denatured protein extraction method according to claim 3, characterized in that: the specific process of the step 3 is as follows:
step 3.1: calculating the dosage of the BPP extracting solution according to the mass of the tissue sample, wherein the dosage of the BPP extracting solution is 7ml of BPP extracting solution used by each gram of the tissue sample;
step 3.2: adding the ground tissue-divided powder into BPP extraction buffer solution according to the calculation mode of 3ml BPP buffer/1g tissue sample, pouring the mixture into the solution, turning upside down and uniformly mixing, and putting the mixture into a refrigerator for hydration at 4 ℃ for 1h for later use;
step 3.3: adding dithiothreitol to a concentration of 5mM before use; extracting for the first time, adding phenylmethylsulfonyl fluoride with the concentration of 1mM before use, mixing uniformly, adding into the tissue powder, mixing uniformly, and performing vortex oscillation for 5min after ultrasonic treatment;
step 3.4: adding equal volume of Tris saturated phenol, wherein the pH value of the Tris saturated phenol is more than 7.8, and vortex mixing for 10min at room temperature;
step 3.5: centrifuging with a centrifuge at 4 deg.C and 12500rpm for 15 min;
step 3.6: preparing a new centrifuge tube, centrifuging, adding phenol, adding an equal volume of BPP extraction buffer solution, adding dithiothreitol to a final concentration of 5mM before the BPP extraction buffer solution is used, mixing uniformly, and vortex mixing at room temperature for 10 min;
step 3.7: centrifuging with a centrifuge at 4 deg.C and 12500rpm for 15 min;
step 3.8: prepare a new centrifuge tube, centrifuge, add phenol, add 5 volumes of pre-cooled saturated ammonium sulfate solution in methanol, precipitate overnight at-20 ℃.
5. The BPP method-based preparation method of sugarcane tissue denatured protein according to claim 4, characterized in that: in the step 3.3, the number of the vortex oscillations after the ultrasound is 30, the time of the vortex oscillations after each time is 10s, and the gap time is 10 s.
6. The method for extracting the sugarcane tissue denatured protein based on the BPP method according to claim 5, characterized in that: the ammonium sulfate methanol solution in step 3.8 is prepared by mixing 6.605g of ammonium persulfate powder and 500ml of methanol.
7. The method for extracting the sugarcane tissue denatured protein based on the BPP method according to claim 6, characterized in that: the specific process of the step 4 is as follows:
step 4.1: subpackaging the mixed solution on ice, and performing centrifugal precipitation by using a centrifugal machine at 4 ℃ and 12500rpm for 15 min;
step 4.2: discarding the supernatant, adding methanol pre-cooled at-20 deg.C into each tube, scattering, resuspending, precipitating, balancing, centrifuging at 4 deg.C for 15min at 12500rpm by use of a centrifuge;
step 4.3: repeating the step 4.2 once, discarding the supernatant, and airing the precipitate;
step 4.4: adding lysate into the precipitate, controlling the concentration of the protein solution at 5-8ug/ul, performing vortex oscillation for 30 times after ultrasonic treatment, wherein the vortex oscillation time is 10s after each time, and the gap time is 10 s;
step 4.5: and (3) centrifuging by using a centrifuge at the temperature of 4 ℃ and the centrifugal speed of 12500rpm for 15min, and collecting supernatant to obtain the denatured whole protein lysate.
8. The method for extracting the sugarcane tissue denatured protein based on the BPP method according to claim 6, characterized in that: and 4.3, air-drying the precipitate, wherein the air-drying degree is based on the fact that the protein block begins to have 1-3mm cracks.
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Application publication date: 20200929 |