CN103278370A - Method sutiable for two-dimensional electrophoresis extraction and separation of total protein of rat and mouse testicular tissue sample - Google Patents
Method sutiable for two-dimensional electrophoresis extraction and separation of total protein of rat and mouse testicular tissue sample Download PDFInfo
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Abstract
The invention discloses a method for two-dimensional electrophoresis extraction and separation of total protein of a rat and mouse testicular tissue sample. The method comprises rat and mouse testicular tissue treatment, a preparation method for a testicular tissue total protein sample, a two-dimensional electrophoresis technology system, gel dyeing and spectrum analysis. The method optimizes a tedious universal two-dimensional electrophoresis technology system with relatively poor stability, greatly reduces time and reagent consumption for finding out conditions, saves experiment cost, and obtains a satisfactory two-dimensional electrophoresis spectrum that is convenient for subsequent analysis. The method is more suitable for the extraction of the rat and mouse testicular tissue protein sample than a universal homogenate extraction method, a lysate extraction method, a lysate extraction/acetone precipitation method at present. The method not only reduces the degradation of the protein but also increases solubility of the protein without increasing cost of the reagent, so that the two-dimensional electrophoresis spectrum with relatively many protein sites, clear horizontal stripes and relatively good repeatability can be obtained.
Description
Technical field
The present invention relates to a kind of extraction and protein science separation method of protein, be specifically related to a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis and extract and the method for separating.
Background technology
Protein is the main big molecule that constitutes the biosome vital movement.Along with the decoding of large number of biological whole genome sequences such as humans and animals and the expansion of functional genome research, life scientist more and more pays close attention to how to carry out the research of proteomics with the pattern of genome research.Proteomics will become one of strategic high ground of maximum strategic resource of new century-human gene fight.At present, the proteome research progress is very rapid in the world, though basic theory or technical method, all constantly progressive and perfect.Because proteomics research is than the more approaching practicality of genomics research, huge market outlook are arranged, become " focus " that each main developed country of west, each transnational pharmacy group competitively drop into.Yet, on the whole the development of proteomics be technology promote also be subjected to technical limitation.The height of its technical method level is depended in the proteomics research success or not to a great extent.Therefore, the investigative technique platform of development high flux, high sensitivity, high accuracy is present and even the main task of quite a while internal protein group research and association area.
The two dimensional gel electrophore-sis technology is one of a kind of core key technology in the proteomics research.Its ultimate principle is first to separate with isoelectric focusing to the isoelectric point based on protein, and second separates with SDS-PAGE to the difference by molecular weight then, finally the protein in the complicated protein mixture at two dimensional surface separately.The connexus spectral technology is identified one by one to the protein that separates simultaneously.Because of its residing critical positions in protein group and medical research, be widely used in that protein is transcribed and posttranscriptional modification research, the comparison of protein group and the interaction between protein, toxicity protein science, the research of cell differentiation apoptosis, the research of mechanism of causing a disease and resistance mechanism, curative effect monitoring, new drug development, cancer research, purity of protein inspection, the small protein purifying, many aspects such as reproductive development biology.Yet a series of shortcomings such as complex steps, instability and muting sensitivity that two dimensional gel electrophore-sis technology itself exists have limited its application greatly.What is more important, the validity of bidirectional electrophoresis technique, reliability and the repeatable preparation that all depends on institute's analyzing proteins sample.For most of animal tissues, the preparation of dielectrophoresis protein sample and whole technique system do not have unified method and program.Most samples all need to grope optimum condition by test of many times, waste time and energy, and experimental cost is higher.
Testing big mouse is the most normal employing model animal, and testis tissue is to relate to protein science in reproductive development and the arrenotoky toxicological study to study requisite material.Therefore, how at big mouse testis tissue, set up a cover effectively, repeatably sample preparation methods and bidirectional electrophoresis technique system, significant to the large-scale promotion application of whole bidirectional electrophoresis technique.The present invention is repeatedly attempting respectively on the big mouse testis protein extraction basis such as the homogenate extraction method that generally adopts at present both at home and abroad, lysate extraction method, lysate extraction/acetone precipitation, lysate extraction/the acetone precipitation of improvement has been proposed, revised the lysate prescription, and optimized whole bidirectional electrophoresis technique system, set up and be fit to big mouse testis dielectrophoresis sample preparation and the method for separating, obtained resolution higher with repeatability electrophoresis pattern preferably.
Summary of the invention
The invention provides a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis and extract and the method for separating, its step is as follows:
(1) will test big mouse and put into the case that fills ether, treat that it suffocates after, be fixed in dissecting pan, cut scrotum, peel off its surrounding tissue, extract testis, and with the ultrapure water flushing, remove tunicle then, weigh;
(2) testis tissue that (1) is handled and the tissue sample protein lysate of precooling are after the ratio of 1:5 is mixed with the mass volume ratio, electronic tissue homogenate instrument 11,000 rpm homogenate, each 10s, interval 1min, 3-5 time continuously, be homogeneous shape suspension until system, whole process sample hose places ice bath all the time;
(3) suspension after (2) processing is at room temperature left standstill 1h, albumen is fully dissolved, then at 4 ℃, centrifugal 1 h of 16000g, draw supernatant then, with the Bradford method measure the protein concentration of supernatant, with supernatant packing 3-4 part by volume, make the protein content of every portion of supernatant be retained to 400-500ug according to measurement result;
(4) with 80% pre-cold acetone mixing of (3) gained supernatant and 5 times of volumes, place 15min-60min for-18--20 ℃;
(5) with (4) treating fluid at 4 ℃, 16, the centrifugal 5min of 000g, supernatant discarded places superclean bench just to put, a gentle wind springing up 10s-30s, protein precipitation sample that must be to be dissolved;
(6) the aquation sample-loading buffer 1ml that gets-20 ℃ of freezing preservations puts in the 1.5ml EP pipe, after the room temperature dissolving, adds 0.01g DTT, 2.5mL Bio-Lyte 4-6,2.5mL Bio-Lyte 5-7, fully mixing takes out 400ml, add (6) and get in the protein precipitation sample, fully dissolving, mixing;
(7) rob the protein sample that (6) are obtained and join in the focusing dish along the edge linearity from left to right with moving liquid, keep the middle sample liquid of groove to link up, the 1-1.2cm distance is respectively stayed at the groove two ends, simultaneously, place salt bridge respectively in the both sides that focus on disc electrode, and soak with the ultrapure water of 10uL, then that aquation is good adhesive tape changes in the focusing dish, and glue faces down, the both positive and negative polarity correspondence, on every adhesive tape, add 2-3mL mineral oil slowly, with the both positive and negative polarity that focuses on groove to good, cover lid;
(8) adhesive tape after (7) are handled is carried out isoelectric focusing, the isoelectric focusing program is set is: the linear 30min of S1 250V, the quick 1h of S2 1000V, the linear 5h of S3 10000V, quick 60,000 volts hours of S4 10000V, the quick random time of S5 500V;
(9) with after the end of (8) focalizer, carry out the adhesive tape balance with adhesive tape level pad I and adhesive tape level pad II respectively immediately, level pad is pressed 5ml/gel and is added, and equilibration time is 12-15min;
(10) adhesive tape after (9) focusing is carried out second immediately to the SDS-PAGE electrophoresis, step is: the acrylamide gel of preparation 12%, every clotting glue coagulant liquid 40-42ml, inject the glass plate interlayer, the space of 1cm is stayed on top, uses the water-saturated n-butanol front cover, keeps the glue face smooth.Polyase 13 0 minute after electrophoresis tank adds 1 * electrophoretic buffer, is connected power supply, with the low current of 16mA/gel/17cm, treat that sample walking out the IPG adhesive tape fully when initial, be condensed into a line after, strengthen electric current again to 24-30mA/gel/17cm, continue 5.5-6 hour, can stop electrophoresis;
(11) gel after taking-up (10) is handled carries out Coomassie brilliant blue G-250 dyeing, and the gel that dyeed is put into the transmission scan instrument and scanned, and charts, and analyzes with dedicated analysis software.
Described big mouse testis tissue sample protein lysate is formulated by following ingredients and method, at first prepare the ultrapure water solution of 7mol/L urea, 2 mol/L thiocarbamides and 4% CHAPS,-80 ℃ of preservations, each time tissue sample centrifugal before, now add 65mmol/L DTT, Bio-Lyte and 1 Inhibitors cocktail of 0.2% 40%.
Described Inhibitors cocktail is produced by U.S. Roche company, and addition is 1 of every 5ml lysate.
Described aquation sample-loading buffer by the thiocarbamide of the urea of 4.2g, 1.52g, 0.4g CHAPS, now add 0.098g DTT, now add Bio-Lyte, 10ml 1% bromophenol blue of 50ml 40%, be settled to 10ml with MilliQ water, be distributed into 10 tubules, every tubule 1ml ,-20 ℃ of refrigerators are preserved.
The adhesive tape that described aquation is good is the prefabricated adhesive tape of IPG with the 17cm of-20 ℃ of freezing preservations, pH 4-7; place 10min in room temperature; remove the protective seam on the prefabricated IPG adhesive tape then, IPG adhesive tape glue is faced down to be placed on the aquation dish sample solution, places 16-20h under the room temperature.
Adhesive tape after the described focusing places the aquation dish, seals with film, can preserve under-20 ℃ 1-7 days.
Beneficial effect of the present invention: protein sample preparation and the bidimensional electrophoretic separation technology of the big mouse testis tissue that (1) the present invention sets up, general bidirectional electrophoresis technique system loaded down with trivial details, less stable is optimized, time (a full experiment time is more than 48 hours) and the reagent consumption of the condition of groping have been significantly reduced, save experimental cost, obtained the satisfied dielectrophoresis collection of illustrative plates of being convenient to subsequent analysis.(2) lysate provided by the invention extracts---the large and small mouse testis tissue sample holoprotein preparation method of acetone precipitation, general homogenate extraction method, lysate extraction method, lysate extraction/acetone precipitation is more suitable for the extraction of big mouse testis tissue protein sample at present, protein sample with the method preparation, under the prerequisite that does not increase reagent cost, not only reduced the degraded of albumen, and increased the dissolubility of albumen, thereby obtain the more and clear horizontal stripe of protein site, repeatability is the dielectrophoresis collection of illustrative plates preferably.(3) large and small mouse testis tissue blood vessel compares comparatively dense; centrifugal below 40000r/min; it is higher to occur salt ionic concentration as the protein sample that still adopts conventional " extract method " to handle through regular meeting; the desalination effect is bad; a large amount of PDs, or first the voltage during to isoelectric focusing does not reach a series of problems such as predeterminated voltage.The present invention is according to the specificity of big mouse testis tissue, through constantly groping, compare repeatedly, continue to optimize, the best approach of finding the preparation protein sample is to analyze pure method (histone extract+acetone precipitation), uses the histone extract of 1:5 to carry out after the cracking with behind the 80% acetone precipitation protein of 1:5, desalination is effective, can obtain the condition that the higher protein sample of purity satisfies isoelectric focusing, voltage can reach predeterminated voltage, obtains comparatively ideal focusing effect.
Accompanying drawing
The rat testis sample two dimensional gel electrophore-sis dyeing collection of illustrative plates that Fig. 1 prepares with the present invention.
The mouse testis tissue sample two dimensional gel electrophore-sis dyeing collection of illustrative plates that Fig. 2 prepares with the present invention.
Embodiment
Set forth embodiments of the present invention below.
Material used in the present invention is: the prefabricated adhesive tape of IPG (U.S. Bio-Rad), and pH 4-7,17cm ,-20 ℃ of refrigerators are preserved; Carrier ampholyte (U.S. Bio-Rad) ,-4 ℃ of refrigerators are preserved; Isoelectric focusing sample-loading buffer (U.S. Bio-Rad), 4 ℃ of refrigerators are preserved; Urea Urea, CHAPS, DTT(Dithiothreitol), bromophenol blue (Bromophenol Blue); Mineral oil (Mineral Oil), SDS, Tris, Iodoacetamide; Low melting-point agarose, glycocoll, acrylamide (Acrylamide); Methylene diacrylamide (Bis), ammonium persulfate (Ammonium Persulfate); TEMED, glycerine, sulphur urine, Bio-Safe TM Coomassie.
Instrument and software that the present invention uses comprise: Y96000 type electronic balance (German Sai Duolisi); Cut tissue refiner in the T18 (German IKA company); Ultra low temperature freezer (Thermo fisher); UV-2100 type spectrophotometer (You Nika (Shanghai) Instr Ltd.); PROTEIN IEF System(U.S. BIO-Rad company); PROTEIN IEF
Xl Cell(U.S. BIO-Rad company); Horizontal shaking table (Changzhou Guohua Electric Appliance Co., Ltd.); UMAX powerlook 2100XL; PDQusetc software(U.S. BIO-Rad company); High speed freezing centrifuge (Sigma company).
The reagent of the present invention's preparation comprises:
(1) cracking night: urea (7M, 4.2g); Thiocarbamide (2M, 1.52g); CHAPS(4%, 0.4g); DTT(65mM, 0.098g, now add); Bio-Lyte 0.2% (w/v) (50ml, 40%, now add); Proteinase(Inhibitor scocktail, 1table);
(2) aquation sample-loading buffer: urea (7M, 4.2g); Thiocarbamide (2M, 1.52g); CHAPS(4%, 0.4g); DTT(65mM, 0.098g, now add); Bio-Lyte(0.2% (w/v), 50ml 40%, now add); Bromophenol blue (0.001%, 10ml 1% bromophenol blue); MilliQ water (being settled to 10ml); Be distributed into 10 tubules, every tubule 1ml ,-20 ℃ of refrigerators are preserved;
(3) adhesive tape level pad mother liquor: urea (6M, 36g); SDS(2%, 2g); PH8.8 Tris-HCl(0.375M, 25ml 1.5M pH8.8 Tris-HCl); Glycerine (20%, 20ml); MilliQ water (being settled to 100ml); Be distributed into 10 pipes, every pipe 10ml ,-20 ℃ of refrigerators are preserved;
(4) adhesive tape level pad I: adhesive tape level pad mother liquor (10ml); DTT(0.2g), abundant mixing, the time spent now joins;
(5) adhesive tape level pad II: adhesive tape level pad mother liquor (10ml); Iodoacetamide (0.25g), abundant mixing, the time spent now joins;
(6) low melting-point agarose sealing liquid: low melting-point agarose (0.5%, 0.5g); Tris(25mM, 0.303g); Glycocoll (192mM, 1.44g); SDS(0.1%, 1ml10% SDS)); Bromophenol blue (0.001%, 100ml 1% bromophenol blue) MilliQ water (being settled to 100ml), heating for dissolving is to clarification, room temperature preservation;
(7) 30% polyacrylamides storage liquid: acrylamide (150g); Methylene diacrylamide (4g); MilliQ water (500ml); After filter paper filtered, 4 ℃ of refrigerators of brown bottle were preserved;
(8) 1.5mol/L Tris alkali pH8.8:Tris alkali (90.75g); MilliQ water (400ml); Transfer pH to 8.8 with 1mol/L HCl, add MilliQ water and be settled to 500ml.4 ℃ of refrigerators are preserved;
(9) 12% SDS:SDS(12g); MilliQ water (100ml); Behind the mixing, room temperature preservation.This condition is groped to draw by experiment;
(10) 10% Ap:Ap(0.1g); MilliQ water (1ml, time spent are dissolved in water); After the dissolving, 4 ℃ of refrigerators are preserved;
(11) 10 * electrophoretic buffers (1 *=25mM Tris, 192mM glycine, 0.1% SDS, pH8.3): Tris alkali (30g); Glycocoll (144g); SDS(10g); MilliQ water (1L); Behind the mixing, room temperature preservation.
The invention provides a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis and extract and the method for separating, its step is as follows:
(1) big mouse testis tissue is handled
Rat to be slaughtered is put into the case that fills ether, treat that it suffocates after, be fixed in dissecting pan.Cut scrotum, peel off its surrounding tissue, extract testis, the stripping tunicle, and place liquid nitrogen rapidly.At last, testis tissue is deposited in ultra low temperature freezer, be used for follow-up specimen preparation.
(2) testis tissue holoprotein sample preparation methods
A disposes protein lysate;
B takes out rat or the mouse testis of peeling off tunicle from ultra low temperature freezer, will organize in right amount and put in the 5mLEP pipe, adds 5 times of volume histone extracts by W/V=1:5 and carries out cracking;
C 11000 rpm homogenate, in each 10 seconds, 3-5 time continuously, certain hour is in case the sample temperature rising is homogeneous shape suspension until system at interval after each homogenate, and sample hose all will place ice bath in the whole process;
The D room temperature leaves standstill 1h, and albumen is fully dissolved, then centrifugal 1h under 4 ℃;
E sucts clearly, through Bradford method side protein concentration, and is divided into 3-4 part, and every part of protein content is 400ug-500ug;
F places 15min-60min for-20 ℃ with the 80% acetone precipitation protein of supernatant with 5 times of volumes, and 4 ℃, 16 then, the centrifugal 5min of 000g, supernatant discarded places superclean bench just to put, a gentle wind springing up 10s-30s, protein precipitation sample that must be to be dissolved;
The albumen of G precipitation is with sample-loading buffer dissolving, sample aquation adhesive tape in the preparation.
(3) bidirectional electrophoresis technique system
A first is to isoelectric focusing (IEF)
From refrigerator, get 1mL aquation sample-loading buffer 2 tubules that configure in advance of-20 ℃ of freezing preservations, put the room temperature dissolving.Add 0.01g DTT in tubule, Bio-Lyte 4-6,5-7(be at pH4-7, the adhesive tape of 17cm) each 2.5mL, fully mixing.Take out 400mL aquation sample-loading buffer then, join in the sample of acetone precipitation desalination, fully get the prefabricated adhesive tape of IPG (17cm, pH 4-7) of-20 ℃ of freezing preservations after the mixing dissolving from refrigerator, placed 10 minutes in room temperature; Rob edge to the left side of groove in the aquation dish and the right linear sample that adds with moving liquid; Each 1cm does not want application of sample at the groove two ends, and middle sample liquid must link up, and guarantees not produce bubble, after all protein examples have all joined in the aquation dish, with the protective seam on the prefabricated IPG adhesive tape of tweezers removal gently; IPG adhesive tape glue faced down place on the aquation dish sample solution, sample solution is got on the plastic support film at the adhesive tape back side, do not make the solution below the adhesive tape produce bubble equally, in case generation bubble, one end of mentioning adhesive tape with tweezers lightly, move up and down adhesive tape, beyond bubble is arrived in adhesive tape.
Put the back well and on every adhesive tape, add 2-3mL mineral oil slowly, prevent evaporation of liquid in the adhesive tape hydration process.Along adhesive tape, make mineral oil drop by drop slowly be added on the plastic support film; Sample carries out in the aquation dish on the aquation of adhesive tape, places 16-20 hour under the room temperature.
Aquation finishes, and shifts adhesive tape.The both sides that at first focus on disc electrode are placed salt bridge respectively, and soak with the ultrapure water of 10uL, then that aquation is good adhesive tape changes in the focusing dish, glue faces down, the both positive and negative polarity correspondence adds 2-3mL mineral oil and the both positive and negative polarity that focuses on groove on the every adhesive tape slowly to good, cover lid, the isoelectric focusing program is set, the beginning isoelectric focusing.Will note voltage, the current conditions of adhesive tape constantly in this process, according to real-time observation, can adjust the focusing process at any time, purpose makes adhesive tape reach the optimal separation effect.
It is the linear 30min(desalination of five steps: the first, 250V that the main process of isoelectric focusing is divided into); The quick 1h(desalination of the second, 1000V); The linear 5h(of the 3rd, 10000V boosts); Quick 60,000 volts hours of the 4th, 10000V (focusing); The quick random time of the 5th, 500V (maintenance); The limiting current of every adhesive tape is set simultaneously.Temperature when (50-70A/ root) and isoelectric focusing (17 ℃).
The balance of B adhesive tape
The adhesive tape that focusing finishes is carried out balance immediately, places the thick filter paper of doing on the table earlier, and the adhesive tape glue that focusing is good faces up and is placed on the dried thick filter paper.With the thick filter paper of another part MilliQ water-soaked, squeeze and remove excessive moisture, keep the moistening of filter paper, prevent from damaging the glue face, directly place then on the adhesive tape, blot mineral oil and unnecessary sample on the adhesive tape gently, and wash adhesive tape gently with MilliQ water.
The adhesive tape that flushing is good moves in the aquation dish, adds 5ml adhesive tape level pad I in the groove of adhesive tape is arranged, and slowly rocks on horizontal shaking table 15 minutes.Subsequently, thoroughly outwell adhesive tape level pad I, and draw unnecessary equilibrium liquid in the aquation dish with filter paper, add 5ml adhesive tape level pad II again, repeat aforesaid operations.Balance begins second to the SDS-PAGE electrophoresis after finishing.
C second is to polyacrylamide gel electrophoresis (SDS-PAGE)
Two of the acrylamide gels of preparation 12% are joined the 80ml gel solution, and every clotting glue 40ml injects the glass plate interlayer respectively with solution, and the space of 1cm is stayed on top, uses the water-saturated n-butanol front cover, keep the glue face smooth.Polyase 13 0 minute.After general gel and the top liquid layering, show gel polymerization substantially, note the preceding sealing with ethanol inspection encapsulating frame of glue, do not produce bubble during glue.After treating that gel solidifies, remove the water-saturated n-butanol on separation gel surface, and go excess liquid between the glass plate of SDS-PAGE polyacrylamide gel top with the filter paper suction.With second being placed on the desktop to gel of handling well, long glass plate is down, short glass plate up, the top of gel is facing to oneself.Agarose sealing liquid and micro-wave oven or metal bath are carried out heating for dissolving.
With 10 * electrophoretic buffer, with 10 times of graduated cylinder dilutions, be made into 1 * electrophoretic buffer, the bubble on the damping fluid surface of rushing.The IPG adhesive tape is shifted out from sample aquation dish, and an end of clamping adhesive tape with tweezers makes the glue face soak the end fully in 1 * electrophoretic buffer.Then adhesive tape glue is faced up and be placed on the long glass plate of gel.
The SDS-PAGE gel that is placed with adhesive tape is transferred on the encapsulating frame, and short glass plate one is facing to oneself.Syringe needle with tweezers, spatula or tack pushes away adhesive tape downwards lightly, makes it to contact fully with polyacrylamide gel glue face.Above gel, add low melting-point agarose sealing liquid then.Note below adhesive tape, not producing any bubble.When pushing away adhesive tape with tweezers, be noted that the support membrane that promotes the gel back side, do not run into the glue face.Placed 5 minutes, low melting-point agarose sealing liquid is thoroughly solidified after.Gel is transferred in the electrophoresis tank.
After electrophoresis tank adds electrophoretic buffer, connect power supply, the low current of using when initial (16mA/gel/17cm) or low-voltage, treat that sample walking out the IPG adhesive tape fully, after being condensed into a line, strengthen electric current (or voltage) again (24mA/gel/17cm), treat to stop electrophoresis when the bromophenol blue indicator reaches bottom margin, generally run 5.5-6h.
(4) dyeing of gel
After electrophoresis finishes, pry open layer glass gently, take out gel, and corner cut is with marking, with washed with de-ionized water gel twice, each 10min is to remove SDS; And it is fixing to carry out spending the night of glue with the acetic acid of 10% the ethanol of volume ratio 1:10 and 10%, adds enough Bio-Safe Commassie stain subsequently and covers gel, and 1h is to spending the night in vibration.Dyeing finishes, and uses the washed with de-ionized water gel under swaying, and color can be deepened gradually behind the 1h; Up to obtaining desirable contrast.
(5) atlas analysis
To dye lustful gel and put into the dedicated scan instrument and scan, drawing, and analyze with dedicated analysis software.
Claims (6)
1. a big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis extracts and the method for separating, and its step is as follows:
(1) will test big mouse and put into the case that fills ether, treat that it suffocates after, be fixed in dissecting pan, cut scrotum, peel off its surrounding tissue, extract testis, and with the ultrapure water flushing, remove tunicle then, weigh;
(2) testis tissue that (1) is handled and the tissue sample protein lysate of precooling are after the ratio of 1:5 is mixed with the mass volume ratio, electronic tissue homogenate instrument 11,000 rpm homogenate, each 10s, interval 1min, 3-5 time continuously, be homogeneous shape suspension until system, whole process sample hose places ice bath all the time;
(3) suspension after (2) processing is at room temperature left standstill 1h, albumen is fully dissolved, then at 4 ℃, centrifugal 1 h of 16000g, draw supernatant then, with the Bradford method measure the protein concentration of supernatant, with supernatant packing 3-4 part by volume, make the protein content of every portion of supernatant be retained to 400-500ug according to measurement result;
(4) with 80% pre-cold acetone mixing of (3) gained supernatant and 5 times of volumes, place 15min-60min for-18--20 ℃;
(5) with (4) treating fluid at 4 ℃, 16, the centrifugal 5min of 000g, supernatant discarded places superclean bench just to put, a gentle wind springing up 10s-30s, protein precipitation sample that must be to be dissolved;
(6) the aquation sample-loading buffer 1ml that gets-20 ℃ of freezing preservations puts in the 1.5ml EP pipe, after the room temperature dissolving, adds 0.01g DTT, 2.5mL Bio-Lyte 4-6,2.5mL Bio-Lyte 5-7, fully mixing takes out 400ml, add (6) and get in the protein precipitation sample, fully dissolving, mixing;
(7) rob the protein sample that (6) are obtained and join in the focusing dish along the edge linearity from left to right with moving liquid, keep the middle sample liquid of groove to link up, the 1-1.2cm distance is respectively stayed at the groove two ends, simultaneously, place salt bridge respectively in the both sides that focus on disc electrode, and soak with the ultrapure water of 10uL, then that aquation is good adhesive tape changes in the focusing dish, and glue faces down, the both positive and negative polarity correspondence, on every adhesive tape, add 2-3mL mineral oil slowly, with the both positive and negative polarity that focuses on groove to good, cover lid;
(8) adhesive tape after (7) are handled is carried out isoelectric focusing, the isoelectric focusing program is set is: the linear 30min of S1 250V, the quick 1h of S2 1000V, the linear 5h of S3 10000V, quick 60,000 volts hours of S4 10000V, the quick random time of S5 500V;
(9) with after the end of (8) focalizer, carry out the adhesive tape balance with adhesive tape level pad I and adhesive tape level pad II respectively immediately, level pad is pressed 5ml/gel and is added, and equilibration time is 12-15min;
(10) adhesive tape after (9) focusing is carried out second immediately to the SDS-PAGE electrophoresis, step is: the acrylamide gel of preparation 12%, every clotting glue coagulant liquid 40-42ml, inject the glass plate interlayer, the space of 1cm is stayed on top, use the water-saturated n-butanol front cover, keep the glue face smooth, polyase 13 0 minute, after electrophoresis tank adds 1 * electrophoretic buffer, connect power supply, with the low current of 16mA/gel/17cm, treat that sample walking out the IPG adhesive tape fully when initial, after being condensed into a line, strengthen electric current again to 24-30mA/gel/17cm, continue 5.5-6 hour, can stop electrophoresis;
(11) gel after taking-up (10) is handled carries out Coomassie brilliant blue G-250 dyeing, and the gel that dyeed is put into the transmission scan instrument and scanned, and charts, and analyzes with dedicated analysis software.
2. a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis according to claim 1 extracts and the method for separating, it is characterized in that described big mouse testis tissue sample protein lysate is formulated by following ingredients and method, at first prepare the ultrapure water solution of 7mol/L urea, 2 mol/L thiocarbamides and 4% CHAPS,-80 ℃ of preservations, each time tissue sample centrifugal before, now add 65mmol/L DTT, Bio-Lyte and 1 Inhibitors cocktail of 0.2% 40%.
3. a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis according to claim 1 extracts and the method for separating, and it is characterized in that described Inhibitors cocktail is produced by U.S. Roche company, and addition is 1 of every 5ml lysate.
4. a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis according to claim 1 extracts and the method for separating, it is characterized in that described aquation sample-loading buffer by the thiocarbamide of the urea of 4.2g, 1.52g, 0.4g CHAPS, now add 0.098g DTT, now add Bio-Lyte, 10ml 1% bromophenol blue of 50ml 40%, be settled to 10ml with MilliQ water, be distributed into 10 tubules, every tubule 1ml ,-20 ℃ of refrigerators are preserved.
5. a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis according to claim 1 extracts and the method for separating; the good adhesive tape that it is characterized in that described aquation is the prefabricated adhesive tape of IPG with 17cm, the pH 4-7 of-20 ℃ of freezing preservations; place 10min in room temperature; remove the protective seam on the prefabricated IPG adhesive tape then; IPG adhesive tape glue faced down to be placed on the aquation dish sample solution, places 16-20h under the room temperature.
6. a kind of big mouse testis tissue sample holoprotein that is suitable for dielectrophoresis according to claim 1 extracts and the method for separating, and it is characterized in that the adhesive tape after the described focusing places the aquation dish, seals with film, can preserve under-20 ℃ 1-7 days.
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| CN2013101424302A Pending CN103278370A (en) | 2013-04-23 | 2013-04-23 | Method sutiable for two-dimensional electrophoresis extraction and separation of total protein of rat and mouse testicular tissue sample |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103884576A (en) * | 2014-04-03 | 2014-06-25 | 华中农业大学 | Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis |
| CN103951730A (en) * | 2014-04-21 | 2014-07-30 | 广西大学 | Method for preparing buffalo testis convoluted seminiferous tubule total protein samples and two-dimensional electrophoresis separation method |
| CN106124599A (en) * | 2016-06-25 | 2016-11-16 | 河南科技大学 | A kind of two-dimensional electrophoresis method for protein and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103884576A (en) * | 2014-04-03 | 2014-06-25 | 华中农业大学 | Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis |
| CN103884576B (en) * | 2014-04-03 | 2017-05-17 | 华中农业大学 | Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis |
| CN103951730A (en) * | 2014-04-21 | 2014-07-30 | 广西大学 | Method for preparing buffalo testis convoluted seminiferous tubule total protein samples and two-dimensional electrophoresis separation method |
| CN106124599A (en) * | 2016-06-25 | 2016-11-16 | 河南科技大学 | A kind of two-dimensional electrophoresis method for protein and application thereof |
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