CN103884576B - Method for extracting and separating whole protein of rat hippocampus, suitable for dimensional electrophoresis - Google Patents

Abstract

The invention discloses a method which is used for extracting and separating whole protein of rat hippocampus and is suitable for dimensional electrophoresis. The method comprises the following steps: (1) placing a rat in frozen artificial cerebrospinal fluid; (2) pouring liquid nitrogen in a mortar for precooling before grinding; (3) standing turbid liquid at room temperature; (4) subpackaging liquid supernatant; (5) adding RB ( rehydration buffer) into protein conglomeration; (6) making a standard curve by 5, 10, 15, 20, 25, 30 and 35 micrograms of BSA (Bull Serum Albumin); (7) loading analysis gel, namely silver staining, with the quantity shown in the specification; (8) taking out a dried gel strip and rewarming at room temperature; (9) adding coverage oil into an isoelectric focusing disk; (10) performing isoelectric focusing treatment; (11) rewarming the treated gel strip; (12) preparing low melting agarose from upper tank liquid; and (13) carrying out coomassie blue staining on the gel and drawing. The method is easy to implement and is suitable for extracting of rat hippocampus tissue protein samples, the operation is simple and convenient, and a dimensional electrophoretogram with more protein spots, clear horizontal stripes and good repeatability is obtained.

Description

A kind of rat hippocampus whole protein suitable for two-dimensional electrophoresis is extracted and detached method

Technical field

The present invention relates to Separation of Proteins identification technology field, is more particularly to a kind of Rat hippocampus sample whole protein Extract and detached method, proteins of hippocampi tissue can be efficiently separated, show protein difference and subsequently identify these eggs White matter, it is adaptable to the related toxicologic study of rat nervous system function and drug screening.

Background technology

Protein is the main executive of organism vital movement.With a large amount of biology whole genome sequences such as humans and animals Decoding and functional genome research expansion, life scientist increasingly pays close attention to the composition of biological vivo protein and its activity Rule.At present, in the world Advances on Proteome is very rapid, no matter rationale or technical method, is all constantly entering Walk and perfect.There is huge market prospect closer to practical than genomics research due to proteomics research, it has also become " focus " that each Main Developed Countries in west, each transnational pharmacy group competitively put into.However, the development of proteomics on the whole It is both that technology is promoted and technical limitations.Proteomics research success or not, is heavily dependent on protein The height of separating and purifying technology method level.Therefore, developing high flux, high sensitivity, the investigative technique platform of high accuracy is The research of present or even quite a while internal protein group and the main task of association area.

Two dimensional gel electrophoresis are a kind of one of core key technologies in proteomics research.Its ultimate principle is First with isoelectrofocusing based on isoelectric point of protein to being separated, and second to the difference for then pressing molecular weight is separated with SDS-PAGE, Finally the protein in complicated protein mixture is separated in two dimensional surface.In combination with mass-spectrometric technique to detached protein by One is identified.Because of its residing critical positions in protein group and medical research, it is widely used in protein transcription and turns Modification research after record, the interaction between the comparison of protein group and protein, toxicity protein science, cell differentiation apoptosis grind Study carefully, the research of mechanism of causing a disease and resistance mechanism, curative effect monitoring, new drug development, cancer research, purity of protein inspection, small protein Many aspects such as purification, reproductive development biology.However, the step of two dimensional gel electrophoresis are present in itself is loaded down with trivial details, unstable Its application is greatly limit with a series of shortcomings such as muting sensitivity.What is more important, the effectiveness of bidirectional electrophoresis technique, reliability Property and repeatability all rely on the preparation of institute's analyzing proteins sample.For most animals tissue, two-dimensional electrophoresis egg The preparation of white sample and whole technical system, without unified methods and procedures.Most of sample is required for by many Optimum condition is groped in secondary test, is wasted time and energy, and experimental cost is higher.

Experimental rat is most frequently with model animal, and Rat hippocampus are to be related to protein science research in neural toxicological study Requisite material.Therefore, how for Rat hippocampus, a set of sample preparation methods effectively, repeatable are set up And two-dimensional electrophoresis system, it is significant to the large-scale promotion application of whole bidirectional electrophoresis technique.The present invention is more Secondary homogenate extraction method, lysate extraction method, the lysate extraction/acetone precipitation for having attempted generally adopting at present both at home and abroad respectively On the basis of the rats such as method are extracted in vain, it is proposed that the lysate extraction-ultrasound of improvement grinds-acetone precipitation method of protein, and excellent Change whole two-dimensional electrophoresis system, established suitable Rat hippocampus dielectrophoresis sample and prepare and detached method, The higher and repeated preferable electrophoresis pattern of resolution is obtained.

The content of the invention

The purpose of the present invention is to there are provided a kind of separation method of Rat hippocampus sample whole protein, and method is easy OK, it is easy to operate, the lysate extraction-ultrasound of the improvement that this experiment is adopted grinds-and acetone precipitation method of protein can effectively keep away Exempt from the iii vitro chemical modification of protein losses and protein, be more suitable for the extraction of Rat hippocampus protein sample, so as to obtain Protein site is more and clear horizontal stripe, repeatability preferably Two-dimensional Gel Electrophoresis.

A kind of rat hippocampus whole protein suitable for two-dimensional electrophoresis is extracted and detached method, and its step is：

（1）, will experiment use SPF level rats（Dead）Afterwards, it is placed in frost artificial cerebrospinal fluid（ACSF）Middle about 1min, takes brain Tissue, is placed in about 30 s in frost ACSF, and hippocampal tissue is isolated on ice, weighs with the Hippocampus in group.

（2）, Rat hippocampus grinding before first pour Liquid nitrogen precooler in mortar, then will（1）The Hippocampus group of process Knit after about 1 g is shredded and be placed in mortar, be rapidly added liquid nitrogen.Grind rapidly after liquid nitrogen not reboiling in mortar, until tissue Become powder, uniformly without obvious granule.

（3）, by step（2）Suspension after process is in room temperature（It is 20-25 DEG C, same as below）Lower standing 1h, fills albumen Divide dissolving, 800 μ L lysates are added in mortar（LB）, fully dissolving（Collect）During 1.5mL EP pipes are transferred to after powder, then Plus 400 μ L LB rinse a mortar, in being transferred to identical EP pipe.Supersound process（80W, opens for 0.8 second, closes within 0.8 second, ultrasound 10 Cooled on ice is put in after lower, 6 times are repeated）, nucleic acid is crushed, 4 DEG C, 12000r/min is centrifuged 20min, collects supernatant.

（4）, by step（3）Supernatant after process is distributed into 4 pipes, often the μ L of pipe about 250, and often pipe adds 1mL acetone -20 DEG C precipitates overnight.In 4 DEG C, 12000r/min is centrifuged 20min to the protein for having precipitated, and removes supernatant, opens lid and lies against Spontaneously dry on clean napkin, you can the protein agglomerate after being processed, -80 DEG C save backup.

（5）, in step（4）200uL RB are added in every tubulin matter group block after process, with pipette tips by protein agglomerate Blow and beat repeatedly after stamp is little（5-20 time）Until protein is fully dispersed.Ultrasonic dissolution assisting, 80W is opened for 0.8 second, is closed within 0.8 second, ultrasound 4 Cooled on ice is put in after secondary（0℃）, repeating 1 time, 4 DEG C after ultrasound, 12000r/min is centrifuged 20min, suctions out supernatant, turns In moving to new EP pipes.

（6）, the BSA that takes 5,10,15,20,25,30,35 μ g make standard curve, the manufacture method of standard curve is as follows：

Take step（4）The μ L of supernatant 2~3 in EP pipes after process, each tube adds 1mL Bradford to be dyeed, whirlpool Rotation vibration 20s so as to light absorption value can be determined after fully mixing, operating interval during measure between two samples should also be 20s or so.Quantitatively carry out at twice, for the first time for preliminary quantitative so as to measured value in the range of standard curve, by sample liquid Absorbance look into standard curve and be calculated after the concentration of each sample, all samples concentration is adjusted to relatively as far as possible, then Carry out second quantitatively, in order to ensure quantitative accuracy, quantitatively all need to make standard curve every time.

（7）, analysis glue（Silver staining）Applied sample amount be the μ g of silver staining 100, mass spectrum glue（Examine dye）Applied sample amount be 1200 μ g.On The composition of sample liquid is：The protein of respective amount, 1%（w/v）DTT、1%（v/v）IPG Buffer, 1 × BPB, RB are to 460 μ L.Root According to step（6）In the concentration data of quantitative gained draw the protein of respective amount,

（8）, from refrigerator take out immobilized ph gradient strip room temperature under rewarming 10min（Water-bath, 23 DEG C）.Step（7）The sample of middle absorption is molten Liquid is homogeneously added into bottom land, and sample-adding is about 22cm.Adhesive tape is taken out, support film is made towards operator, from acidic terminal（There is word end）Start, Gently tear coverlay, down, acidic terminal is put into adhesive tape in the groove of aquation disk towards top glue surface.Put well and added after adhesive tape 1.2mL covers oil in moisturizing on film is supported, it is 20 DEG C to adjust indoor temperature, allows adhesive tape swelling overnight（10~12h）, record glue The number of bar.

（9）, in isoelectrofocusing disk add cover oil（Every swimming lane adds 6mL）.Take out step（8）Adhesive tape after process And the oil of the covering in adhesive tape is blotted, glue surface is upward put into adhesive tape in isoelectrofocusing disk.Paper gasket is sheared, each pad adds 75 μ L Milli Q water carries out moistening, and paper gasket is put into the two ends of glue surface, edge and glue surface justified margin, loads onto battery lead plate, selects Just can bring into operation program after adhesive tape quantity.One to isoelectrofocusing program is：S1 stp 300V 0:30Hr；S2 stp 700V 0:30Hr；S3 stp 1500V 1:30Hr；S4 grd 9000V 3:00Hr；S5 stp 9000V 4:00Hr, entirely focused on Cheng Suoyong total voltages：52KVh.

(10), by step（9）After isoelectrofocusing has been processed, adhesive tape is taken out, blot the covering oil on surface, glue surface is upward Adhesive tape is put in balance pipe, -20 DEG C of preservations.

（11）, take out step（10）The adhesive tape of process carries out rewarming（Water-bath, 23 DEG C）, add the washing of 10mL or so deionization Fall the covering oil of adhesive tape surface.Every balance pipe adds 8mL balance liquids to be balanced, and first adds 100mM DTT balance 15min, 250mM IAA are added to balance 15min after clean balance liquid at once, IAA wants lucifuge when balancing.

（12）, with tank liquor configure 0.8% (w/v) low melting-point agarose 40mL, 1.5% (w/v) plain agar sugar 80mL, be put in after scorification in foam box be incubated it is standby.The liquid in glass plate glue surface is outwelled, the liquid of residual is inhaled with filter paper It is dry, draw eutectic agarose and seal whole glue surface.It is rapid to take out step（11）Adhesive tape after middle balance, connects and start after electrode SDS-PAGE electrophoresis electrophoresis.Can stop electrophoresis to after glue when BPB runs out of two.The program of two to SDS-PAGE electrophoresis is：S1 2W/gel 45min；S2 17W/gel, until bromophenol blue goes to bottom, about 4:30h

（13）, take out step（12）Gel after process carries out Coomassie brilliant G-250 dyeing, has dyeed gel and has been put into Penetrate in scanner and scan, drawing, and be analyzed with dedicated analysis software.

The Ginding process of described Rat hippocampus is as follows：First Liquid nitrogen precooler is poured in mortar before grinding, then will Hippocampal tissue is placed in mortar after shredding, and is rapidly added liquid nitrogen.Grind rapidly after liquid nitrogen not reboiling in mortar, until tissue Become powder.

Described ultrasonication processing routine is as follows：80W, opens for 0.8 second, 0.8 second close, ultrasound 10 times after be put in it is cold on ice But, repeat 6 times, crush nucleic acid, 4 DEG C, 12000r/min is centrifuged 20min, collects supernatant.

Described step（9）In one be to isoelectrofocusing program：S1 stp 300V 0:30Hr

S2 stp 700V 0:30Hr

S3 stp 1500V 1:30Hr

S4 grd 9000V 3:00Hr

S5 stp 9000V 4:00Hr

Total voltage used by whole focusing：52KVh.

Described step（12）In the program of two to SDS-PAGE electrophoresis be：S1 2W/gel 45min； S2 17W/gel Until bromophenol blue goes to bottom, about 4:30h.

Described step（12）In two to SDS-PAGE electrophoresis encapsulating liquid compositions and content it is as follows：

The present invention compared with prior art, with advantages below and effect：

（1）The protein sample of the big mice Rat hippocampus that the present invention sets up is prepared and bidimensional electrophoretic separation technology, will The general two-dimensional electrophoresis system of loaded down with trivial details, less stable is optimized, and greatly reduces the time of the condition of groping（One Secondary complete test period more than 48 hours）With reagent consumption, experimental cost is saved, obtain satisfied subsequent analysis of being easy to Two-dimensional Gel Electrophoresis.

（2）The lysate of the improvement that the present invention is provided extracts-and ultrasound grinds the-Rat hippocampus of acetone precipitation protein Sample whole protein preparation method, relatively general at present homogenate extraction method, lysate extraction method, lysate extraction/acetone precipitation Method is more suitable for the extraction of big mice Rat hippocampus protein sample, the protein sample prepared with the method, is not increasing reagent On the premise of cost, not only reduce the degraded of albumen, and increased the dissolubility of albumen, so as to obtain protein site it is more and Clear horizontal stripe, repeatability preferably Two-dimensional Gel Electrophoresis.

（3）The protein sample that conventional " extracting solution method " is processed often occurs that salt ionic concentration is higher, and desalination effect is bad, A large amount of PDs, or first to voltage during isoelectrofocusing do not reach predeterminated voltage a series of problems, such as.Basis of the present invention The specificity of Rat hippocampus, through constantly groping, compares repeatedly, continues to optimize, and discovery prepares hippocampal protein sample The best approach is ultrasonic extraction（Supersound extraction+acetone precipitation）, the electricity such as can obtain the higher protein sample of purity to meet The condition of focusing, voltage can reach predeterminated voltage, obtain comparatively ideal focusing effect.

We extract and separate energy on obtained Two-dimensional Gel Electrophoresis using this method to Rat hippocampus whole protein Detect more than 2950 protein spotses.

Description of the drawings

Fig. 1 is that a kind of Rat hippocampus sample two dimensional gel electrophore- sises dye collection of illustrative plates.

Fig. 2 is that a kind of Rat hippocampus sample two dimensional gel electrophore- sises dye collection of illustrative plates.

The Two-dimensional Gel Electrophoresis obtained using this technology possess higher resolution and repeatability.Can be seen that from figure The technology can effectively go the removal of impurity, reduce protein degradation, increase protein solubility, and focusing effect is good, manifests on gel Protein spotses it is many, and clearly.

Specific embodiment

Embodiments of the present invention are described below.

Material used in the present invention is：The prefabricated adhesive tape of IPG（U.S. Bio-Rad）, pH4-7,17cm, -20 DEG C of refrigerators guarantors Deposit；Carrier ampholyte（U.S. Bio-Rad）, -4 DEG C of Refrigerator stores；Isoelectrofocusing sample-loading buffer（U.S. Bio-Rad）, 4 DEG C Refrigerator store；Carbamide Urea, CHAPS, DTT（Dithiothreitol）, bromophenol blue（Bromophenol Blue）；Mineral oil （Mineral Oil）、SDS、Tris、Iodoacetamide；Low melting-point agarose, glycine, acrylamide （Acrylamide）；Methylene diacrylamide（Bis）, ammonium persulfate（Ammonium Persulfate）；TEMED, glycerol, sulfur Urea, Bio-Safe TM Coomassie.

The instrument and software that the present invention is used includes：Y96000 type electronic balances（German Sai Duolisi）；Cut group in T18 Knit refiner（German IKA companies）；Ultra cold storage freezer（Thermo fisher）；UV-2100 type spectrophotometers（You Nika（On Sea）Instrument Ltd.）；PROTEIN IEF System（BIO-Rad companies of the U.S.）；PROTHIN IEF Cell（The U.S. BIO-Rad companies）；Horizontal shaker（Changzhou Guohua Electric Appliance Co., Ltd.）；UMAX powerlook 2100XL；PDQusetc software（BIO-Rad companies of the U.S.）；High speed refrigerated centrifuge（Sigma companies）, Ultrasound Instrument（The new sesame biotechnology stock in Ningbo Part company limited Ningbo Scientz Biotechnology Co., the n of LTD JY96- II）.

The reagent that the present invention is prepared includes：

Alklysis buffer(LB)

Reagent	Final concentration
		Tris	20mM
Thiourea (FW 76.12)	2M
		Urea (FW 60.06)	7M
CHAPs (FW 64.89)	2%(W/V)
		Double distilled water

- 80 DEG C of preservations

rehydration buffer stock(RB)

Reagent	Final concentration
		Urea (Amersham)	7M
Thiourea (Amersham)	2M
		CHAPs(Usb)	4%(W/V)
Double distilled water

- 80 DEG C of preservations

Bradford

Reagent	Final concentration
		Methanol (Tianjin great Mao chemical reagent factories)	23.75mL
Phosphoric acid (Tianjin great Mao chemical reagent factories)	50 mL
		G250(Bio Basic Inc.)	50mg
Double distilled water	To 500 mL

Bromophenol blue solution

Reagent	Final concentration
		Bromophenol blue（Bio Basic Inc.）	1%
Tris-base（Amresco）	50mM
		Double distilled water

Room temperature preservation

30%T,2.6%C Monomer stock solution

Reagent	Final concentration
		Acylamide （Amresco）	30%
N,N’-methylenebisacrylamide (Sigma)	0.8%
		Double distilled water

0.2 μm of membrane filtration, 4 DEG C keep in dark place

4× Resolving gel buffer

Reagent	Final concentration
		Tris-base（Amresco）	1.5M
HCl (Guangzhou Chemical Reagent Factory)	To pH8.8
		Double distilled water

0.2 μm of membrane filtration, 4 DEG C of preservations

10% SDS

Reagent	Final concentration
		SDS（Promega）	10%
Double distilled water

0.2 μm of membrane filtration, room temperature preservation

10× SDS electrophoresis buffer

Reagent	Final concentration
		SDS（Promega）	1%
Tris-base（Amresco）	250mM
		Glycine（Amresco）	1.92M
Double distilled water

SDS equilibration buffer

Reagent	Final concentration
		Tris-HCl (pH8.8)（Amresco）	50mM
Urea(Amersham)	6M
		Glycerol (Amersham)	30%(v/v)
SDS（Promega）	2% (w/v)
		Bromophenol blue（Bio Basic Inc.）	0.002%(w/v)
Double distilled water

- 20 DEG C of preservations

A kind of Rat hippocampus sample whole protein for being suitable for two-dimensional electrophoresis is extracted and detached method, and its step is such as Under：

（1）Rat hippocampus process：

SPF level rats are used in experiment（Dead）Afterwards, it is placed in frost artificial cerebrospinal fluid（ACSF）Middle about 1min, takes brain group Knit, be placed in about 30 s in frost ACSF, hippocampal tissue is isolated on ice, weigh with the Hippocampus in group.

（2）Hippocampal tissue whole protein sample preparation methods

A, configuration protein lysate（LB）：20 mM Tris, 2 M Thiourea,7 M Urea, 2% w/v CHAPs 。

B, first will pour Liquid nitrogen precooler in mortar before appropriate hippocampal tissue about 1g grindings, then shred hippocampal tissue After be placed in mortar, be rapidly added liquid nitrogen.Grind rapidly after liquid nitrogen not reboiling in mortar, until tissue becomes powder, It is even without obvious granule.

C, suspension is stood at room temperature 1h, albumen is fully dissolved, 800 μ L LB are added in mortar, it is fully molten Solution（Collect）During 1.5mL EP pipes are transferred to after powder, then 400 μ L LB are added to rinse a mortar, in being transferred to identical EP pipe. Supersound process（80W, opens for 0.8 second, 0.8 second close, ultrasound 10 times after be put in 0 DEG C of cooled on ice, repeat 6 times）, broken nucleic acid, 4 DEG C, 12000r/min is centrifuged 20min, collects supernatant.

D, supernatant are distributed into 4 pipes, often the μ L of pipe about 250, and often pipe adds -20 DEG C of precipitates overnights of 1mL acetone.Precipitate Protein in 4 DEG C, 12000r/min is centrifuged 20min, removes supernatant, open lid lie against it is natural on clean napkin It is dried, you can the protein agglomerate after being processed, -80 DEG C save backup.

E, add in every tubulin matter group block 200uL RB, with pipette tips by protein agglomerate stamp it is little after blow and beat repeatedly it is straight It is fully dispersed to protein.Ultrasonic dissolution assisting, 80W is opened for 0.8 second, 0.8 second close, ultrasound 4 times after be put in cooled on ice（0℃）, then weigh Multiple 1 time, 4 DEG C after ultrasound, 12000r/min is centrifuged 20min, supernatant is suctioned out, in being transferred to new EP pipes.

F Jing Bradford methods survey protein concentration, and are divided into 3-4 parts, and every part of protein content is 400ug-500ug；

Often pipe adds 1mL -20 DEG C of precipitates overnights of acetone to G.In 4 DEG C, 12000r/min is centrifuged the protein for having precipitated 20min, removes supernatant, opens lid and lies against natural drying on clean napkin, you can the albumen matter group after being processed Block, -80 DEG C save backup.

It is prepared by adhesive tape：

Aquation disk is taken out, level is adjusted standby.Rewarming 10min under immobilized ph gradient strip room temperature is taken out from refrigerator.After drawing centrifugation Sample supernatant is added in the groove of aquation disk, and sample solution is homogeneously added into into bottom land, and sample-adding is about 22cm.Adhesive tape is taken out, is made Film is supported towards operator, from acidic terminal（There is word end）Start, coverlay of gently tearing, down, acidic terminal is towards top by glue for glue surface Bar is put in the groove of aquation disk, and operating process will avoid producing bubble.Put well and add after adhesive tape 1.2mL coverings oil in support film Upper moisturizing, it is 20 DEG C to adjust indoor temperature, allows adhesive tape swelling overnight（10~12h）, record the number of under strip.

Isoelectrofocusing：

Start pre-cooling, takes out battery lead plate, adds in isoelectrofocusing disk and covers oil（Every swimming lane adds 6mL）.Take out glue Bar simultaneously blots the oil of the covering in adhesive tape（Covering in oil may have the sample liquid not being sucked in gel）, glue surface is upward put into adhesive tape In isoelectrofocusing disk.Paper gasket is sheared, each pad adds 75 μ L Milli Q water to carry out moistening, and paper gasket is put into the two of glue surface End, edge and glue surface justified margin, load onto battery lead plate, select the program that just can bring into operation after adhesive tape quantity.

Isoelectrofocusing program is：S1 stp 300V 0:30Hr

S2 stp 700V 0:30Hr

S3 stp 1500V 1:30Hr

S4 grd 9000V 3:00Hr

S5 stp 9000V 4:00Hr

Total voltage used by whole focusing：52KVh

The preservation of adhesive tape：

After running through isoelectrofocusing, adhesive tape is taken out, blot the covering oil on surface, adhesive tape is put into balance pipe by glue surface upward In, -20 DEG C of preservations.

Second to polyacrylamide gel electrophoresis（SDS-PAGE）Encapsulating.

Dress encapsulating device：

Black rubber cushion is loaded into encapsulating device, centering is compressed.Glass plate is closed into tight, until glass plate can be at desktop station It is vertical.Load thick plastic base plate in the encapsulating mouth one side of encapsulating device, fillet upward, loads glass plate, refills thick plastic base plate, successively After glass plate is all installed, load thin plastic base plate, fill up whole encapsulating device.Deionization is crowded with the groove of encapsulating device plate Water, loads onto sponge strip, notices that sponge strip should be flattened, and loads onto plate, clip on folder, upright to place encapsulating device, adjusts level standby.

With liquid encapsulating：

12.5%SDS-PAGE gel formulas

Glue is configured according to formula table, AP is stirred while being slowly added to as TEMED, it is to avoid localized rich is spent It is high and condense.Glue is set to flow down along the thick plastic base plate on encapsulating mouth inclined-plane during encapsulating, action is uniform, fills to liquid level to glass Under the parting bead of glass plate both sides till 4mm.It is rapid to add water-saturated n-butanol to flatten liquid level in glue surface, plus fashionable make pipette tips against glass The slow stroke of long slab is uniformly added into, and every glue adds 4mL n-butyl alcohol, and solidification is overnight.

Prepare glass plate：

Tear glass plate open, the broken glue of glass plate outer surface is cleaned up, deionized water is rinsed once, is noted not during cleaning Tap water is allowed to be flushed to glue surface.Deionized water is rinsed glass plate is put on shelf after glue surface 6 times, is squeezed some deionized waters and is existed Moisturizing in glue surface.

Tank liquor in configuration：

Electrophoresis tank circulating water pipe is connected with the water pipe of cooler, 4L deionized waters are added in electrophresis apparatuses water tank, plugged The plug of electrophoresis tank circulating pump, after waiting air to drain, with graduated cylinder 450mL10 × electrophoresis liquid is measured, and adds 50mL deionized waters, is delayed It is slow to pour in electrophoresis tank, be made into 1 × lower tank liquor.The plug of cooler is plugged, cooler is opened and is started pre-cooling.Measure 240mL 10 × electrophoresis liquid, add water to 1.2L configurations 2 × upper tank liquor, it is standby after stirring.

Adhesive tape is balanced：

Balance liquid be separately added into it is standby after 100mM DTT and 250mM IAA stir, IAA stir when answer lucifuge.Take out Adhesive tape carries out rewarming, adds 10mL or so deionized water to wash the covering oil of adhesive tape surface off.Every balance pipe adds 8mL balance liquids It is balanced, first adds DTT balance 15min, add IAA to balance 15min after clean balance liquid at once, IAA wants lucifuge when balancing.

Two to electrophoresis：

With low melting-point agarose 40mL, the 1.5% plain agar sugar 80mL of tank liquor configuration 0.8%, bubble is put in after scorification It is incubated in foam box standby.The liquid in glass plate glue surface is outwelled, the liquid of residual is blotted with filter paper, draw eutectic agarose envelope Firmly whole glue surface.Rapidly adhesive tape is taken out from balance pipe, somewhat rinsed in the graduated cylinder for load onto tank liquor, adhesive tape is put into into glass In glass plate, acidic terminal is pressed onto two to Jiao Jiaomianchu with ruler towards left placement adhesive tape, makes adhesive tape lower end be in close contact two to glue glue Face, is careful not to allow adhesive tape lower end to bottle up bubble.

Put well successively glass plate is fitted in lower groove after adhesive tape, tiltable during glaze plate is put into, do not allow glass plate lower end Bottle up bubble.Install and load onto after glass plate groove, then add plain agar sugar to seal gap at the gap of groove and glass plate and prevent Only leak electricity in vertical direction.Tank liquor dilutes one times and is configured to maximum scale with tank liquor on lightly adding in upper groove After lower tank liquor, it is added in lower groove until upper and lower tank liquor face is equal, closes the lid, connects and start after electrode electrophoresis.When BPB runs out of Two can stop electrophoresis to after glue.

Two to the program of electrophoresis is：S1 2W/gel 45min

S2 17W/gel go to bottom until bromophenol blue, and about 4:30h

The dyeing of gel：

Stop electrophoresis to after glue when BPB runs out of two, groove and lower groove, start to tear glue open in taking-up.With plastics ruler by glass Plate is tilted, and is careful not to break the wire electrode of lower groove, and wash clean glass plate selects the dyeing of respective number according to the number of adhesive tape Disk（Dyeing disk need to first mark number, and be initially charged 250mL fixatives）.Glass plate is stuck up out with plastics ruler, gently removes glue Bar, dials the agarose of glue surface, and cuts a little angle in the acidic terminal of glue surface with plastics ruler.Deionized water rinses glass plate Lower surface and glue surface, and squeezing bottle head is extended into flushing under glue, make glue somewhat depart from glass plate, by glass plate left-hand thread to dyeing disk On, allow glue slowly to depart from glass plate by gravity and fall in dyeing disk.Dyeing disk is placed on horizontal shaker and is vibrated, room temperature is fixed More than 2h.

Silver staining

Step	Solution	Consumption	Time
				It is fixed	Ethanol	100mL	More than 2h
	Glacial acetic acid	25mL
					Anhydrous sodium acetate	0.17g
	Plus distilled water is to 250mL
				Sensitization	Ethanol	75mL	60min
	Sodium thiosulfate（NaS2O3·5H2O）	0.788g
					Plus distilled water is to 250mL
Rinsing	Distilled water 250mL		4×10min
				Silver staining	Silver nitrate	0.625g	60min
	Plus distilled water is to 250mL
				Rinsing	Distilled water		2×1min
Colour developing	Sodium carbonate	6.25g	8min
					Formaldehyde（37%w/v）	100uL
	Plus distilled water is to 250mL
				Terminate	Na2EDTA·H2O	3.65g	45min
	Plus distilled water is to 250mL
				Rinsing	Distilled water 250mL		2×30min

Examine dye

Step	Solution	Consumption	Time
				It is fixed	Ethanol	100mL	More than 2h
	Glacial acetic acid	25mL
					Anhydrous sodium acetate	0.17g
	Plus distilled water is to 250mL
				Rinsing	Distilled water 250mL		4×10min
Examine dye	G-250	0.3g	More than 6h
					Ammonium sulfate	25g
	Phosphoric acid	25mL
					Methanol	50mL
	Plus distilled water is to 250mL
				Rinsing	Distilled water 250mL		2×1min

The scanning and preservation of gel：

First scanner is wiped clean before scanning, some water are sprayed on glass, then gel is transferred on scanner, will be cut The one end at angle is placed on lower-left Angle Position, it is ensured that the direction of every glue scanning is identical, and scan pattern is the perspective scanning of 256 GTGs, Resolution is 200dpi.For the mass spectrum glue for examining dye, need on the glass of scanner one wash clean of pad glass plate, then by glue Put and scan on a glass, it is ensured that glue surface it is clean, in order to avoid introduce pollutant during Mass Spectrometric Identification.The gel preservative film for scanning through 4 DEG C of preservations are placed in after wrapping.

Graphical analyses

Analysis software is ImageMaster 2D platinum 5.0 (GE).

The inventive method is optimized loaded down with trivial details, less stable general two-dimensional electrophoresis system, subtracts significantly Lack time and the reagent consumption of the condition of groping, saved experimental cost, obtain the satisfied two-way electricity for being easy to subsequent analysis Swimming collection of illustrative plates, relatively current general homogenate extraction method, lysate extraction/acetone precipitation are more suitable for Rat hippocampus albumen sample The extraction of product, on the premise of reagent cost is not increased, not only reduces the degraded of albumen, and increased the dissolving of albumen Property, horizontal stroke common in two-way gel, longitudinal grin phenomenon are effectively reduced, more and clear so as to obtain protein site, repeatability is preferably Two-dimensional Gel Electrophoresis.

Claims (1)

1. a kind of rat hippocampus whole protein suitable for two-dimensional electrophoresis is extracted and detached method, and its step is：

(1) after experiment is processed with SPF levels rat, 1min in frost artificial cerebrospinal fluid is placed in, takes cerebral tissue, be placed in frost ACSF Middle 30s, isolates on ice hippocampal tissue, weighs with the Hippocampus in group；

(2) first pour Liquid nitrogen precooler in mortar before the grinding of Rat hippocampus, hippocampal tissue 1g of step (1) process is cut Be placed in after broken in mortar, add liquid nitrogen, liquid nitrogen is not ground after reboiling in mortar, until tissue becomes powder, uniformly without Obvious granule；

(3) suspension after step (2) process is stood at room temperature 1h, makes protein dissolution, add 800 μ L to crack in mortar Liquid, during 1.5mL EP pipes are transferred to after dissolved powders, then adds 400 μ L LB to rinse a mortar, in being transferred to identical EP pipe, surpasses Sonication, crushes nucleic acid, and 4 DEG C, 12000r/min is centrifuged 20min, collects supernatant；

(4) supernatant after step (3) process is distributed into into 4 pipes, often the μ L of pipe 250, often pipe adds -20 DEG C of precipitations of 1mL acetone Overnight, in 4 DEG C, 12000r/min is centrifuged 20min to the protein for having precipitated, and goes supernatant, opening lid to lie against clean Spontaneously dry on napkin, the protein agglomerate after being processed, -80 DEG C save backup；

(5) step (4) process after every tubulin matter group block in add 200uL RB, with pipette tips by protein agglomerate stab it is little after Piping and druming is put in cooled on ice up to protein dispersibility, ultrasonic dissolution assisting after ultrasound, repeats 1 time, 4 DEG C after ultrasound, 12000r/ Min, is centrifuged 20min, supernatant is suctioned out, in being transferred to new EP pipes；

(6) BSA for taking 5,10,15,20,25,30,35 μ g makes standard curve, takes supernatant in the EP pipes after step (4) is processed 2~3 μ L, each tube adds 1mL Bradford to be dyeed, and vortex oscillation 20s determines light absorption value after mixing, determine two samples Operating interval between product is 20s, is quantitatively carried out at twice, is quantitatively calculated the concentration of each sample for preliminary for the first time, then Carry out second quantitatively, for quantitative accuracy, quantitatively all make standard curve every time；

(7) applied sample amount for analyzing glue is the μ g of silver staining 100, and the applied sample amount of mass spectrum glue is 1200 μ g, and the composition of sample solution is：Respective amount Protein, 1%w/vDTT, 1%v/vIPG Buffer, 1 × BPB, the μ L of RB to 460, according to quantitative gained in step (6) Concentration data draws the protein of respective amount；

(8) from rewarming 10min, water-bath under refrigerator taking-up immobilized ph gradient strip room temperature, 23 DEG C, the sample solution drawn in step (7) is uniform Ground adds bottom land, is loaded long 22cm, takes out adhesive tape, from the beginning of acidic terminal, coverlay of tearing, and down, acidic terminal is towards top for glue surface Adhesive tape is put in the groove of aquation disk, is put well and add after adhesive tape 1.2mL coverings oil in moisturizing on film is supported, adjust indoor temperature For 20 DEG C, adhesive tape swelling is allowed overnight, record the number of under strip；

(9) add in isoelectrofocusing disk and cover oil, every swimming lane adds 6mL, take out the adhesive tape after step (8) is processed and blot Covering oil in adhesive tape, glue surface is upward put into adhesive tape in isoelectrofocusing disk, shears paper gasket, and each pad adds 75 μ L Milli Q water carries out moistening, and paper gasket is put into the two ends of glue surface, edge and glue surface justified margin, loads onto battery lead plate, selects adhesive tape Bring into operation program after quantity, and one to isoelectrofocusing program is：S1stp 300V 0:30Hr；S2stp 700V 0:30Hr； S3stp 1500V 1:30Hr；S4grd 9000V 3:00Hr；S5stp 9000V 4:00Hr, total electricity used by whole focusing Pressure：52KVh, KVh are kilovolt hour, and Vh, volt hour represents integration of the voltage to action time；

(10) after step (9) isoelectrofocusing has been processed, adhesive tape is taken out, blots the covering oil on surface, glue surface is upward by adhesive tape In being put into balance pipe, -20 DEG C of preservations；

(11) taking out the adhesive tape of step (10) process carries out rewarming, and water-bath, adds 10mL deionized waters to wash adhesive tape surface off by 23 DEG C Covering oil, every balance pipe adds 8mL balance liquids to be balanced, first plus 100mM DTT balance 15min, clean balance liquid 250mM IAA balance 15min, IAA is added to want lucifuge when balancing afterwards；

(12) the low melting-point agarose 40mL of 0.8%w/v, the plain agar sugar 80mL of 1.5%w/v, scorification are configured with tank liquor After be put in foam box and be incubated standby, outwell the liquid in glass plate glue surface, the liquid of residual is blotted with filter paper, draws eutectic Agarose seals whole glue surface, takes out the adhesive tape after balance in step (11), connects beginning SDS-PAGE electrophoresis, BPB after electrode Run out of two and stop electrophoresis to after glue, two to the program of electrophoresis is：S1 2W/gel 45min；S2 17W/gel, until bromophenol blue Go to bottom, 4:30h；

(13) taking out the gel after step (12) is processed carries out Coomassie brilliant G-250 dyeing, has dyeed gel and is put into transmission and has swept Retouch in instrument and scan, drawing, and be analyzed with dedicated analysis software；

The ultrasonication processing routine of step (3) is as follows：80W, opens for 0.8 second, 0.8 second close, ultrasound 10 times after be put in cooled on ice, Repeat 6 times, crush nucleic acid, 4 DEG C, 12000r/min is centrifuged 20min, collects supernatant；

Two to SDS-PAGE electrophoresis encapsulating liquid compositions and content are as follows in described step (12)：