CN104761614A - Method for extracting water buffalo sperm protein in steps - Google Patents

Abstract

The invention discloses a method for extracting water buffalo sperm protein in steps. Effects of dissolving high, medium and low hydrophilic proteins are achieved by splitting sperm cells in three steps in sequence through three lysate. The liquid supernatant obtained by splitting three times is mixed to obtain total proteins of water buffalo sperms. The method is high in extracting efficiency, less in degradation, high in separating stability and low in cost, a conventional one-step protein extracting method can be replaced, the defect that the proteins are incompletely extracted is made up in the one-step process, the proteins with different hydrophobic properties and the proteins with extreme physical and chemical properties of the sperms are completely extracted, so that a purpose of showing more protein information is achieved, and the sperm protein preparation problem is solved. The total proteins obtained by the method are suitable for two-way electrophoretic analysis, and the proteins extracted by the method are shown more and less in trailing according to atlas analysis on the proteins obtained by a three-step process through a two-way electrophoretic technology.

Description

The method of step by step arithmetic buffalo sperm protein matter

Technical field

The invention belongs to animal reproduction biological technical field, particularly relate to a kind of method of step by step arithmetic buffalo sperm protein matter.

Background technology

At present, the Main Bottleneck run in the proteomics research of buffalo sperm is: during proteins extraction, and a large amount of protein loss or degraded cause proteomics data flux not enough.Cause the major cause of this problem to be, conventional protein one-step extraction can not dissolved cell protein, particularly hydrophobic protein completely, and its physico-chemical property is special, is difficult to be obtained completely by single stage method.And sperm is a kind of cell of specialization; its cytolemma forms primarily of hydrophobic membrane protein; for the protection of the nucleus of sperm inside and DNA from external injury; therefore; spermatid film has the ability of the common lysate of stronger opposing; the lysate of common single stage method is difficult to make sperm dissolve completely and discharge protein, and cause in the experiment in later stage, proteome data presents not exclusively.Therefore, a kind of high efficiency sperm protein matter extracting method is developed imperative.

Summary of the invention

The technical problem to be solved in the present invention is to provide that a kind of extraction efficiency is high, the method for the step by step arithmetic buffalo sperm protein matter of less degradation, to substitute traditional single stage method extraction method, the protein realizing the different hydrophobic proteins of sperm and extreme physico-chemical property all extracts, and reaches the object presenting greater protein matter information.

For solving the problems of the technologies described above, the present invention by the following technical solutions: the method for step by step arithmetic buffalo sperm protein matter, adopts three kinds of lysates to divide three steps cracking spermatid successively; Three kinds of lysates are respectively by following formulated:

Lysate 1---40mM trimethylamino methane, pH 8.5,1mM proteinase inhibitor PMSF;

Lysate 2---7M urea, the dithiothreitol (DTT) of 100mM, 3-[(3-courage amidopropyl) the dimethylammonio]-1-propanesulfonic acid salt of 4%, 1mM proteinase inhibitor PMSF;

Lysate 3---4M urea, 2M thiocarbamide, 2%3-[(3-courage amidopropyl) dimethylammonio]-1-propanesulfonic acid salt, 2mM tri-normal-butyl phosphorus, 1mM proteinase inhibitor PMSF.

The method of above-mentioned step by step arithmetic buffalo sperm protein matter, in being undertaken by following operation: first add lysate 1 in spermatid, collected by centrifugation supernatant; Residue precipitation adds lysate 2, centrifuging and taking supernatant; Residue precipitation adds lysate 3, centrifuging and taking supernatant; By the supernatant mixing obtained after three breakup, obtain the gross protein of buffalo sperm.

The method of above-mentioned step by step arithmetic buffalo sperm protein matter, comprises the following steps:

(1) in spermatid precipitation, 200 pl of lysis liquid 1 are added, in-20 DEG C of multigelations 3-5 time, ice bath fragmentation 5 minutes in Ultrasonic Cell Disruptor, ultrasonic time is 2 seconds, and the intermittent time is 2 seconds; Add DNA lytic enzyme and RNA lytic enzyme 4 DEG C again to place 30 minutes; In refrigerated centrifuge, within centrifugal 10 minutes, get supernatant in 12000 revs/min, precipitation is used for next step process;

(2) by precipitation 40mM trimethylamino methane remaining in step (1), pH8.5, cleans 2 times, add the lysate 2 of 200 microlitres, vibration mixing, after 5 minutes, gets supernatant in centrifugal 10 minutes in 12000 revs/min in refrigerated centrifuge, and precipitation is used for next step process;

(3) by precipitation 40mM trimethylamino methane remaining in step (2), pH8.5, cleans 2 times, add the lysate 3 of 200 microlitres, intense oscillations mixes 5 minutes, in refrigerated centrifuge, within centrifugal 10 minutes, get supernatant in 12000 revs/min, and precipitation is used for next step process;

(4) above step (1) is merged into a pipe to the supernatant that (3) obtain, add the acetone of 6 times of volumes in-20 DEG C of precipitates overnight, centrifugal 10 minutes in 12000 revs/min in refrigerated centrifuge, abandon supernatant, precipitation is the gross protein of buffalo sperm.

For the problem that buffalo sperm protein matter extraction efficiency is low, we have established a kind of method of step by step arithmetic buffalo sperm protein matter, adopt three kinds of lysates to divide three steps cracking spermatid successively, reach the effect to high, medium and low hydrophilic protein matter step-wise dissolution.In three kinds of lysates, lysate 1 can dissolve lower hydrophobic protein containing trimethylamino methane (Tris-HCl), after ultrasonication, cellularstructure is destroyed, the intracytoplasmic water soluble protein of sperm is discharged into outside born of the same parents, and these protein can effectively be dissolved in lysate 1; Lysate 2 uses urea as denaturing agent, and urea is strong denaturant, is interacted by interionic, can interrupt in protein molecule and intermolecular various chemical bond, destroy the secondary of protein, three grades and quaternary structure, make protein more easily molten; The denaturing agent more active than urea-denatured effect is introduced in lysate 3---thiocarbamide three normal-butyl phosphorus, disulfide linkage can be closed into sulfydryl by it, avoids protein again to form secondary structure.By the supernatant mixing obtained after three breakup, obtain the gross protein of buffalo sperm.Extraction efficiency of the present invention is high, less degradation, separating stable degree is high, expense is low, traditional protein one-step extraction can be substituted, compensate for the incomplete defect of proteins extraction in single stage method, the protein realizing the different hydrophobic proteins of sperm and extreme physico-chemical property all extracts, reach the object presenting greater protein matter information, solve sperm protein matter and prepare problem.The gross protein that this method obtains is applicable to Two-dimensional Electrophoresis Analysis, carries out atlas analysis discovery through bidirectional electrophoresis technique to the protein that three-step approach obtains, and the protein that this method is extracted presents more, and hangover is few.

Accompanying drawing explanation

Fig. 1 is the Two-dimensional Gel Electrophoresis that the method applying step by step arithmetic buffalo sperm protein matter of the present invention obtains.

Fig. 2 is the Two-dimensional Gel Electrophoresis that the method applying traditional onestep extraction buffalo sperm protein matter obtains.

Embodiment

1. raw material and reagent

Buffalo sperm takes from Guangxi autonomous region breed improvement station; Retentive force immobilized ph gradient strip (IPGs, immobiline pHgradient DryStrip) is purchased from GE company; Dithiothreitol (DTT) (DL-dithiothreitol, DTT) and three normal-butyl phosphorus (tributylphosphine, TBP) are purchased from Amersco company.IPGphor isoelectric focusing electrophoresis instrument is purchased from GE company.

Wherein, the three kinds of lysates used in following step by step arithmetic are respectively by following formulated:

Lysate 1---40mM trimethylamino methane (Tris-HCl), pH 8.5,1mM proteinase inhibitor PMSF;

Lysate 2---7M urea (Urea), the dithiothreitol (DTT) (DTT) of 100mM, 3-[(3-courage amidopropyl) the dimethylammonio]-1-propanesulfonic acid salt (CHAPS) of 4%, 1mM proteinase inhibitor PMSF;

Lysate 3---4M urea (Urea), 2M thiocarbamide (Thiourea), 2%3-[(3-courage amidopropyl) dimethylammonio]-1-propanesulfonic acid salt) (CHAPS), 2mM tri-normal-butyl phosphorus (TBUP), 1mM proteinase inhibitor PMSF.

2. the concrete steps of the method for step by step arithmetic buffalo sperm protein matter

(1) 200 microlitre Buffalo Semens are centrifugal 15 minutes in 3000 revs/min, discard the impurity such as the refining of supernatant, obtain precipitation and be spermatid, use phosphate buffered saline buffer detergent daughter cell 3 times;

(2) in spermatid precipitation, 200 pl of lysis liquid 1 are added, in-20 DEG C of multigelations 3-5 time, ice bath fragmentation 5 minutes in Ultrasonic Cell Disruptor, ultrasonic time is 2 seconds, and the intermittent time is 2 seconds; Add DNA lytic enzyme (final concentration: 0.01 microgram/microlitre) and RNA lytic enzyme (0.05 microgram/microlitre) 4 DEG C again to place 30 minutes, in order to remove DNA and RNA; In refrigerated centrifuge, within centrifugal 10 minutes, get supernatant in 12000 revs/min, precipitation is used for next step process;

(3) by precipitation 40mM trimethylamino methane (Tris-HCl remaining in step (2), pH8.5) clean 2 times, add the lysate 2 of 200 microlitres, vibration mixing is after 5 minutes, in refrigerated centrifuge, within centrifugal 10 minutes, get supernatant in 12000 revs/min, precipitation is used for next step process;

(4) by precipitation 40mM trimethylamino methane (Tris-HCl remaining in step (3), pH8.5) clean 2 times, add the lysate 3 of 200 microlitres, intense oscillations mixes 5 minutes, in refrigerated centrifuge, within centrifugal 10 minutes, get supernatant in 12000 revs/min, precipitation is used for next step process;

(5) above step (2) is merged into a pipe to the supernatant that (4) obtain, add the acetone of 6 times of volumes in-20 DEG C of precipitates overnight, centrifugal 10 minutes in 12000 revs/min in refrigerated centrifuge, abandon supernatant, precipitation is the gross protein of buffalo sperm, by precipitation two-dimensional electrophoresis sample-loading buffer (buffer formulation: 5M urea, 2M thiocarbamide, 2%3-[(3-courage amidopropyl) dimethylammonio]-1-propanesulfonic acid salt, 40mM dithiothreitol (DTT), 0.5%pH 3-10 amphotericeledrolyte sample-loading buffer) redissolve after, namely can be used for two-dimensional electrophoresis experiment.

3. two-dimensional electrophoresis

3.1 isoelectrofocusing: a. takes out IPG adhesive tape, balances under the condition of room temperature; B. take out appropriate hydrating fluid, treat that it at room temperature dissolves; C. clean adhesive tape groove, and dried up with Blowing drum; D. protein concentration and applied sample amount per sample, determines sample loading volume, adds in new EP pipe; E. with rifle, sample mix liquid is added to the acidic terminal of adhesive tape groove, tears the supporting film of IPG adhesive tape, and discard, then the alkalescence end of adhesive tape is clamped with tweezers, make the acidic terminal of adhesive tape aim at the acidic terminal of adhesive tape groove, inclination adhesive tape groove, the flowing that adhesive tape follows liquid is slowly put down; F. add appropriate covering oil on adhesive tape, covered, then cover lid, be placed on isoelectric focusing electrophoresis instrument; G., isoelectrofocusing parameter and working procedure are set, as shown in table 1:

Table 1 isoelectrofocusing parameter and working procedure (13cm)

The balance of 3.2 adhesive tape: get appropriate level pad from the refrigerator of-20 DEG C, be placed in thaw at RT, then prepares balance liquid I and balance liquid II.Balance I: add 1%DTT in level pad; Balance II: add in 4%IAA and level pad.Then the IPG adhesive tape completing isoelectrofocusing is taken out, be placed in equilibration tube, then pour balance liquid I into, equilibration tube is put on shaking table, carry out the balance of 15min.First time carefully outwells the liquid in pipe, adds balance liquid II, continue to balance 15min on shaking table after balancing and terminating.After second time balance terminates, add dd H ₂o washing twice.

3.3 second to SDS-PAGE: the gel liquid needed for preparation, and solution formula is as shown in table 2;

Table 2 separation gel formula (12%, 12.5%)

3.4 second to electrophoresis: the one end of clamping IPG adhesive tape with clean tweezers, after the one side that adhesive tape is smooth touch offset plate, unclamps tweezers, and the bottom of IPG adhesive tape is connected with the tight of gel bubble-free.Then appropriate sepharose is added in Jiao Ding.According to electrophoresis chamber service requirements, the offset plate sealing top is put into electrophoresis chamber.Then the 1X electrophoretic buffer of specified amount is added.Electrophoretic parameters is set, carries out electrophoresis, be positioned at apart from when being about 1mm at the bottom of glue until tetrabromophenol sulfonphthalein band, stop electrophoresis.

3.5 silver medal dyes

Silver staining method dyes, as shown in table 3:

Table 3 silver dye program

3.6 IMAQ

The gel of silver dye will be completed, to be smoothly put on scanner, and make bubble-free between the two.Open scanning software, carry out prescan, then region suitable on gel lived by frame, formally scans.Image as shown in Figure 1.

Adopt traditional single stage method to extract buffalo sperm gross protein, result as shown in Figure 2 simultaneously.

Relatively two width Two-dimensional Gel Electrophoresis can obviously find, the gross protein that multistep processes of the present invention is extracted presents more protein spot, the relatively uniform whole region being distributed in gel, and particularly low-abundance albumen can be separated better, figure spectral resolution is higher, better quality.But extract on protein pattern in the single stage method of Fig. 2, gel both sides are almost without protein site, and especially on the right of gel, and the some comparatively dense of centre, close together between points, collection of illustrative plates exists more conditions of streaking.Both compare discovery, and the protein example that multistep processes is extracted is more suitable for the Two-dimensional Electrophoresis Analysis of carrying out buffalo sperm.

Claims (3)

1. a method for step by step arithmetic buffalo sperm protein matter, is characterized in that employing three kinds of lysates divide three steps cracking spermatid successively; Described three kinds of lysates are respectively by following formulated:

Lysate 1---40mM trimethylamino methane, pH 8.5,1mM proteinase inhibitor PMSF;

Lysate 2---7M urea, the dithiothreitol (DTT) of 100mM, 3-[(3-courage amidopropyl) the dimethylammonio]-1-propanesulfonic acid salt of 4%, 1mM proteinase inhibitor PMSF;

Lysate 3---4M urea, 2M thiocarbamide, 2%3-[(3-courage amidopropyl) dimethylammonio]-1-propanesulfonic acid salt, 2mM tri-normal-butyl phosphorus, 1mM proteinase inhibitor PMSF.

2. the method for step by step arithmetic buffalo sperm protein matter according to claim 1, is characterized in that being undertaken by following operation: first in spermatid, add lysate 1, collected by centrifugation supernatant; Residue precipitation adds lysate 2, centrifuging and taking supernatant; Residue precipitation adds lysate 3, centrifuging and taking supernatant; By the supernatant mixing obtained after three breakup, obtain the gross protein of buffalo sperm.

3. the method for step by step arithmetic buffalo sperm protein matter according to claim 1, is characterized in that comprising the following steps:

(1) in spermatid, add 200 pl of lysis liquid 1, in-20 DEG C of multigelations 3-5 time, broken 5 minutes of ice bath in Ultrasonic Cell Disruptor, ultrasonic time is 2 seconds, and the intermittent time is 2 seconds; Add DNA lytic enzyme and RNA lytic enzyme 4 DEG C again to place 30 minutes; In refrigerated centrifuge, within centrifugal 10 minutes, get supernatant in 12000 revs/min, precipitation is used for next step process;

(2) by precipitation 40mM trimethylamino methane remaining in step (1), pH8.5, cleans 2 times, add the lysate 2 of 200 microlitres, vibration mixing, after 5 minutes, gets supernatant in centrifugal 10 minutes in 12000 revs/min in refrigerated centrifuge, and precipitation is used for next step process;

(3) by precipitation 40mM trimethylamino methane remaining in step (2), pH8.5, cleans 2 times, add the lysate 3 of 200 microlitres, intense oscillations mixes 5 minutes, in refrigerated centrifuge, within centrifugal 10 minutes, get supernatant in 12000 revs/min, and precipitation is used for next step process;

(4) above step (1) is merged into a pipe to the supernatant that (3) obtain, add the acetone of 6 times of volumes in-20 DEG C of precipitates overnight, centrifugal 10 minutes in 12000 revs/min in refrigerated centrifuge, abandon supernatant, precipitation is the gross protein of buffalo sperm.