阅读说明：本技术 能够阻滞高盐诱导小鼠升高的基因-atf4 (Raised gene-the ATF4 of inducing mouse with high salt can be blocked ) 是由 陈利国 陈伟豪 刘天浩 方梅霞 肖雅 周永红 何玲 袁静 许清芸 陈旭东 陶文聪 于 2019-08-07 设计创作，主要内容包括：本发明公开了一种能够阻滞高盐诱导小鼠升高的基因-ATF4,涉及医药技术领域,该能够阻滞高盐诱导小鼠升高的基因-ATF4中ATF4在高盐诱导小鼠高血压中的作用,发现敲除或敲低ATF4可阻滞高盐诱导小鼠的血压升高。敲除或敲低ATF4基因对血管具有保护作用。(The invention discloses one kind can block the raised gene-ATF4 of inducing mouse with high salt, it is related to pharmaceutical technology field, this can block effect of the ATF4 in inducing mouse hypertension with high salt in the raised gene-ATF4 of inducing mouse with high salt, and discovery, which knocks out or strike low ATF4, can block the blood pressure raising of inducing mouse with high salt.Low ATF4 gene pairs blood vessel is knocked out or struck with protective effect.)

1. one kind can block the raised gene-ATF4 of inducing mouse with high salt, it is characterised in that: described to block high Salt treatment Raised gene-the ATF4 of mouse includes experimental animal: ATF4 gene knockout strikes low C57BL/6J mouse and is purchased from Nanjing animal mould Formula research institute, wild type C57BL/6J mouse are purchased from Nanfang Medical Univ's animal center, raise in Ji'nan University's experimental animal pipe SPF grades of reason center barrier environment；Animal packet: ATF4 gene knockout strikes low C57BL/6J Strains of Mouse 18, and wild type is small Mouse 18, be divided into gene knockout+normal diet group (KO-NS:6 only) ,+8% high salt diet group of gene knockout (KO-HS:12 is only), Wild type+normal diet group (WT-NS:6 is only) ,+8% high salt diet group of wild type (WT-HS:12 is only)；Modeling method: WT-NS Group and KO-NS group give normal mouse forage feed, and it is high that WT-HS group and KO-HS group give Nantong Te Luofei company customizes 8% Salt forage feed, during which free diet, free water, feed 28 days altogether；Mouse aorta pectoralis is taken to be Western within 29th day The expression of blot detection ATF4 gene.

2. according to claim 1 can block the raised gene-ATF4 of inducing mouse with high salt, it is characterised in that: described The operating procedure of the raised gene-ATF4 of inducing mouse with high salt can be blocked:

One, blood pressure detecting

Training mouse 3 days before the formal experiment of beginning, 3 points of every afternoon measures the blood pressure of mouse；When repeatability is good for measurement result, And when the systolic pressure standard deviation very little of measuring assembly, start formal experiment, in formally measurement blood pressure, adjusts mouse in advance Platform temperature is 37 degrees Celsius, carries out pressure calibration and pressure calibration verifying, and check tail sleeve pressure leakage (during measurement weekly Carry out a pressure calibration and pressure calibration verifying), when mouse platform temperature stabilizes to 37 degrees Celsius, act it is soft will be small Mouse, which is placed on to warm up on platform, stands 5 minutes or so, so that mouse is adapted to measurement environment, keeps when measurement measurement room quiet, reduce It is anxious before mouse test, mouse is placed in mouse fixing device later, by mousetail by tail sleeve, passes through V-type On the sensor of trench bottom, and the tail of mouse is fixed on platform with medical proof fabric, rubber sleeve should cover when measurement Rat-tail root (apart from rat-tail root 0.5CM or so), rubber sleeve should push fixation；Measurement rubber sleeve position all Ying Xiangtong every time, It opens Blood Pressure Analysis program to start to measure, take mouse from animal platform away after measurement, mouse It is no more than 30 minutes on test platform, primary every group of the pressure value of measurement before formal experiment, and every 4 after formal experiment Its blood pressure of measurement, measures 7 times altogether, and whole cycle is 28 days；3 points of mouse every afternoon is placed in solid during formal experiment Determine device 10 minutes, and the operation of every step is completed by same operator；

Two, total protein extracts

1, add 10 μ l PMSF (100mM) and 10 μ l Cocktail by 1ml lysate, shake up be placed on ice (PMSF to shake up to It can just be mixed with lysate when nodeless mesh)；

2, tissue need to add liquid nitrogen grinding, add lysate (100mg tissue) of the 600 μ l containing PMSF, in cracking 30min on ice, be It cracks cell sufficiently and wants frequent waggle；

3,14000rpm is centrifuged 5min at 4 DEG C after having cracked.(opening centrifuge pre-cooling in advance)；

4, the supernatant packing after centrifugation is shifted in clean centrifuge tube and is put in -80 DEG C of preservations；

5, the protein solution for having extracted sample and 5 × sample-loading buffer are boiled 10min, is slowly restored room temperature by 4: 1 mixing Afterwards, it is slightly centrifuged, is put in -20 DEG C of preservations；

Three, SDS-PAGE electrophoresis

1, before gel electrophoresis, every 1 × electrophoretic buffer of Kong Junyong is cleaned, and 1 × electrophoretic buffer is added in upper and lower layer electrophoresis tank, Buffer liquid level requires more than loading hole top in the slot of upper layer；

2, according to the loading of sample sequence and loading volume successively loading；

3, electrophoresis: after 80V constant pressure electrophoresis to bromophenol blue to separation gel, 120V constant pressure electrophoresis to the rigid plastic emitting bottom of bromophenol blue stops；

Four, Protein transfer

1, pvdf membrane methanol pre-processes 3~5 seconds, puts to transferring film buffer and infiltrates half an hour；

2, gel is taken out, is put to filter paper, gel is formed and transfers stack layer, filter paper, gel, pvdf membrane, filter paper, gel turn Print " sandwich " structure as stack layer.This operation must completely remove bubble；

3, transfer folder is placed by cathode and anode directions；

4, under cryogenic conditions, 100V constant pressure 116 minutes；

Five, immunoblotting analysis

1. take out hybond membrane, TBST rinse 5min, 1 time；

2.5% skimmed milk power solution room temperature is closed 1 hour；

3.TBST washes film 10min, and 1 time；

4. 4 DEG C of suitable primary antibody diluted concentration overnight；

5.TBST washes film 10min, and 4 times；

6. 37 DEG C of incubation 1h of corresponding secondary antibody diluent；

7.TBST washes film 10min, and 4 times；

8. hybond membrane is placed on a transparent plastic sheet, it is careful not to make film dry；

9. chemiluminescence luminous substrate to be equably added to the surface of film with a clean pipettor, and make to react lasting 5min；

10. sucking the extra substrate solution of film surface with the filter paper that kit provides, put to magazine；

11. development.

Technical field

The present invention relates to pharmaceutical technology field, in particular to one kind can block the raised gene-of inducing mouse with high salt ATF4。

Background technique

High blood pressure is a kind of characterized by angiosthenia increases, can be with the complete of the target organ damages such as the heart, brain, kidney and blood vessel Body disease.The occurrence and development of control high blood pressure have become important topic urgently to be resolved.Vascular endothelial cell has height Flourishing endoplasmic reticulum is intracellular membranous type/secreted protein synthesis organelle, is correctly folded in regulatory protein matter, calcium homeostasis Play a significant role with Apoptosis etc., is the important subcellular organelle for maintaining function of vascular endothelium.When a variety of stimulus The aggregation for inducing unfolded protein matter in endoplasmic reticulum leads to unbalance, the destruction endoplasmic reticulum stable state of calcium ion and redox reaction, Corresponding signal path will be activated, series reaction, referred to as er stress are caused.

The ERS of appropriateness can be by adjusting rapidly intracellular Ca²⁺Balance, protein folding modification and Apoptosis, to tie up The stable state for holding interior environment makes cell constantly adapt to the change of environment and play protective effect, but continues, excessive er stress It can cause the generation of disease.It has proven to be taken part in as the vascular endothelial cell obstacle that ERS causes various as caused by injury of blood vessel Pathophysiological process and related disease, such as hypertension, atherosclerosis, cerebral apoplexy.Recent studies suggest that knocking out people's blood vessel Endothelial cell er stress marker protein ATF4 can reduce vasoconstrictive factor ET-1mRNA and protein expression level, it was demonstrated that ATF4 is the activator of ET-1.The above result of study prompt, the excessive activation of er stress may be intravascular in high blood pressure It plays an important role in chrotoplast dysfunction.Not yet some researches show that ATF4 may participate in the formation of hypertension at present.

Summary of the invention

Technical problem to be solved by the invention is to provide one kind can block the raised gene-of inducing mouse with high salt ATF4, the mouse blood pressure that can block high Salt treatment increase.

To achieve the above object, the present invention provides technical solution below:

It includes experimental animal that this, which can block the raised gene-ATF4 of inducing mouse with high salt: ATF4 gene knockout strikes low C57BL/6J mouse is purchased from Nanjing zootype research institute, and wild type C57BL/6J mouse is purchased from Nanfang Medical Univ animal The heart is raised in center SPF grades of barrier environment of Ji'nan University's management of laboratory animal；Animal packet: ATF4 gene knockout strikes low C57BL/6J Strains of Mouse 18, wild-type mice 18, it is divided into gene knockout+normal diet group (KO-NS:6 is only), clpp gene Except+8% high salt diet group (KO-HS:12 is only), wild type+normal diet group (WT-NS:6 is only) ,+8% high salt diet of wild type Group (WT-HS:12 is only)；Modeling method: WT-NS group and KO-NS group give normal mouse forage feed, WT-HS group and KO-HS group 8% forage feed with high salt of Nantong Te Luofei company customization is given, during which free diet, free water, fed 28 days altogether；29th It takes mouse aorta pectoralis to do the expression that Western blot detects ATF4 gene.

1. the operating procedure that the raised gene-ATF4 of inducing mouse with high salt can be blocked:

One, blood pressure detecting

Training mouse 3 days before the formal experiment of beginning, 3 points of every afternoon measures the blood pressure of mouse；When measurement result repeatability Very well, and when the systolic pressure standard deviation very little of measuring assembly, start formal experiment, in formally measurement blood pressure, adjust in advance Saving mouse platform temperature is 37 degrees Celsius, carries out pressure calibration and pressure calibration verifying, and checks tail sleeve pressure leakage (the measurement phase Between carry out a pressure calibration and pressure calibration verifying weekly), when mouse platform temperature stabilizes to 37 degrees Celsius, movement is soft Mouse be placed on to warm up on platform stand 5 minutes or so, so that mouse is adapted to measurement environment, measurement room peace kept when measurement It is quiet, it is anxious before reducing mouse test, mouse is placed in mouse fixing device later, mousetail is passed through into tail sleeve, The tail of mouse is fixed on platform on the sensor of V-groove bottom, and with medical proof fabric, rubber sleeve when measurement It should cover at rat-tail root (apart from rat-tail root 0.5CM or so), rubber sleeve should push fixation；Measurement rubber sleeve position is all answered every time It is identical, it opens Blood Pressure Analysis program and starts to measure, take mouse from animal platform after measurement It walks, mouse is no more than 30 minutes on test platform, primary every group of the pressure value of measurement before formal experiment, and is formally testing It afterwards every the blood pressure of measurement in 4 days, measures 7 times altogether, whole cycle is 28 days；3 points of mouse every afternoon puts during formal experiment It is placed at fixator 10 minutes, and the operation of every step is completed by same operator；

Two, total protein extracts

1, add 10 μ l PMSF (100mM) and 10 μ l Cocktail by 1ml lysate, shake up and be placed on ice that (PMSF will shake It is even to nodeless mesh when can just be mixed with lysate)；

2, tissue need to add liquid nitrogen grinding, add lysate (100mg tissue) of the 600 μ l containing PMSF, crack on ice 30min wants frequent waggle to crack cell sufficiently；

3,14000rpm is centrifuged 5min at 4 DEG C after having cracked.(opening centrifuge pre-cooling in advance)；

4, the supernatant packing after centrifugation is shifted in clean centrifuge tube and is put in -80 DEG C of preservations；

5, the protein solution for having extracted sample and 5 × sample-loading buffer are boiled into 10min, slow recovery room by 4: 1 mixing Wen Hou is slightly centrifuged, and is put in -20 DEG C of preservations；

Three, SDS-PAGE electrophoresis

1, before gel electrophoresis, every 1 × electrophoretic buffer of Kong Junyong is cleaned, and 1 × running buffer is added in upper and lower layer electrophoresis tank Liquid, buffer liquid level requires more than loading hole top in the slot of upper layer；

2, according to the loading of sample sequence and loading volume successively loading；

3, electrophoresis: after 80V constant pressure electrophoresis to bromophenol blue to separation gel, 120V constant pressure electrophoresis to the rigid plastic emitting bottom of bromophenol blue Only；

Four, Protein transfer

1, pvdf membrane methanol pre-processes 3~5 seconds, puts to transferring film buffer and infiltrates half an hour；

2, taking-up gel, is put to filter paper, formation gel transfer stack layer, filter paper, gel, pvdf membrane, and filter paper coagulates Glue transfers " sandwich " structure as stack layer.This operation must completely remove bubble；

3, transfer folder is placed by cathode and anode directions；

4, under cryogenic conditions, 100V constant pressure 116 minutes；

Five, immunoblotting analysis

1. take out hybond membrane, TBST rinse 5min, 1 time；

2.5% skimmed milk power solution room temperature is closed 1 hour；

3.TBST washes film 10min, and 1 time；

4. 4 DEG C of suitable primary antibody diluted concentration overnight；

5.TBST washes film 10min, and 4 times；

6. 37 DEG C of incubation 1h of corresponding secondary antibody diluent；

7.TBST washes film 10min, and 4 times；

8. hybond membrane is placed on a transparent plastic sheet, it is careful not to make film dry；

9. chemiluminescence luminous substrate to be equably added to the surface of film with a clean pipettor, and continue reaction 5min；

10. sucking the extra substrate solution of film surface with the filter paper that kit provides, put to magazine；

11. development.

Be using the beneficial effect of above technical scheme: this can block in the raised gene-ATF4 of inducing mouse with high salt Effect of the ATF4 in inducing mouse hypertension with high salt, discovery, which knocks out or strikes low ATF4, can block the blood pressure liter of inducing mouse with high salt It is high.Low ATF4 gene pairs blood vessel is knocked out or struck with protective effect.

Detailed description of the invention

A specific embodiment of the invention is described in further detail with reference to the accompanying drawing.

Fig. 1 is the Technology Roadmap that can block the raised gene-ATF4 of inducing mouse with high salt；

Fig. 2 is that the blood pressure that can block the raised gene-ATF4 of inducing mouse with high salt shrinks display diagram；

Fig. 3 is ATF4 albumen relative expression quantity；

Fig. 4 is ELISA statistical results chart.

Specific embodiment

The invention will now be described in detail with reference to the accompanying drawings can block the preferred reality of the raised gene-ATF4 of inducing mouse with high salt Apply mode.

Fig. 1, Fig. 2, Fig. 3 and Fig. 4 show the specific reality that the present invention can block the raised gene-ATF4 of inducing mouse with high salt Apply mode:

In conjunction with Fig. 1, Fig. 2, Fig. 3 and Fig. 4, experimental animal: ATF4 gene knockout strikes low C57BL/6J mouse and is purchased from Nanjing Zootype research institute, wild type C57BL/6J mouse are purchased from Nanfang Medical Univ's animal center, and raising is tested in Ji'nan University SPF grades of the care of animal center barrier environment.

Animal packet: ATF4 gene knockout strikes low C57BL/6J Strains of Mouse 18, and wild-type mice 18.It is divided into base Because of knockout+normal diet group (KO-NS:6 is only) ,+8% high salt diet group of gene knockout (KO-HS:12 is only), wild type+normal drink Food group (WT-NS:6 is only) ,+8% high salt diet group of wild type (WT-HS:12 is only).

Modeling method: WT-NS group and KO-NS group give normal mouse forage feed, and WT-HS group and KO-HS group give south 8% forage feed with high salt of Tong Teluofei company customization, during which free diet, free water, feed 28 days altogether；It takes within 29th day small Mouse aorta pectoralis does the expression of Western blot detection ATF4 gene.

Antibody: ATF4 Antibody (11815S), GAPDH Loading Control antibody (51332S), Goat Anti-Mouse IgG antibody (14709S) is purchased from CST company, the U.S..

Laboratory apparatus and consumptive material: BP-2000 SERIES II Blood Pressure Analysis System is purchased from Visitech Systems company, microplate reader (3530910449) are purchased from silent winged generation that (Shanghai) Instrument Ltd. of match, vertically Electrophoresis tank (VE180) and electrophoretic blotting groove (VE186) are purchased from Shanghai Tian Neng Science and Technology Ltd., electrophoresis apparatus (BG-Power 600i) be purchased from Beijing Baijing Biotechnology Co., Ltd., shaking table (TS-1) is purchased from Fuhua Instrument Ltd., Jintan City, at a high speed from Scheming (TGL-16R) is purchased from Zhujiang River unexpected rival, and Ultrasound Instrument cell crushing instrument (JY92-IIN) is purchased from the new sesame biotechnology share in Ningbo Co., Ltd, camera obscura and dark room lamp (AX-II) are purchased from Yuehua Medical Equipment Factory Co., Ltd., Guangdong.

Other materials and chemical reagent: mouse feed is purchased from Nantong Te Luofei feed technology Co., Ltd, medical X-ray glue Piece (XBT-1) is purchased from Kodak, luminescent solution IMMOBILON WESTERN CHEMILUM HRP SUBSTRATE (WBKLS0500) MILLIPORE company, pvdf membrane Immobilon-P Transfer Membrane (IPVH00010) purchase are purchased from In MILLIPORE company, Cocktail (P8340) is purchased from Sigma, inhibitors of phosphatases (KGP602) and BCA method protein content Detection kit (KGPBCA) is purchased from Development Co., Ltd, Nanjing Keygen Biotech, PageR μ Ler Prestained Protein Ladder (26617) and albumen pre-dyed Marker (SM0672) are purchased from Thermo company, RIPA lysate and primary antibody dilution (P0013) it is purchased from the green skies Bioisystech Co., Ltd in Shanghai, Tris Base, glycine, skimmed milk power, which are purchased from the good science and technology of prestige, to be had Limit company, SDS, glycerol, Beta- mercaptoethanol, ammonium persulfate are purchased from Beijing prosperity Bioisystech Co., Ltd, ancient cooking vessel state, BPB purchase In vast Tyke, acrylamide 30% (Lot1108) is purchased from Shen energy lottery industry biotechnology company, ethyl alcohol, methanol, anhydrous sulfurous acid Sodium, sodium thiosulfate, glacial acetic acid are purchased from Guangzhou board chemical reagent, and Tween-20 is purchased from the limited public affairs of raw work bioengineering (Shanghai) Department, contrasting power are purchased from Tianjin century extensive and profound in meaning commerce and trade Co., Ltd.

Technology Roadmap is as shown in Figure 1.

This can block the operating procedure of the raised gene-ATF4 of inducing mouse with high salt:

One, blood pressure detecting

Training mouse 3 days before the formal experiment of beginning, 3 points of every afternoon measures the blood pressure of mouse；When measurement result repeatability Very well, and when the systolic pressure standard deviation very little of measuring assembly, start formal experiment.In formally measurement blood pressure, adjust in advance Saving mouse platform temperature is 37 degrees Celsius, carries out pressure calibration and pressure calibration verifying, and checks tail sleeve pressure leakage (the measurement phase Between carry out weekly a pressure calibration and pressure calibration verifying).When mouse platform temperature stabilizes to 37 degrees Celsius, movement is soft Mouse be placed on to warm up on platform stand 5 minutes or so, so that mouse is adapted to measurement environment, measurement room peace kept when measurement It is quiet, it is anxious before reducing mouse test.Mouse is placed in mouse fixing device later, mousetail is passed through into tail sleeve, The tail of mouse is fixed on platform on the sensor of V-groove bottom, and with medical proof fabric.Rubber sleeve when measurement It should cover at rat-tail root (apart from rat-tail root 0.5CM or so), rubber sleeve should push fixation；Measurement rubber sleeve position is all answered every time It is identical, it opens Blood Pressure Analysis program and starts to measure.Mouse is taken from animal platform after measurement It walks, mouse is no more than 30 minutes on test platform.Primary every group of the pressure value of measurement before formal experiment, and formally testing It afterwards every the blood pressure of measurement in 4 days, measures 7 times altogether, whole cycle is 28 days；3 points of mouse every afternoon puts during formal experiment It is placed at fixator 10 minutes, and the operation of every step is completed by same operator.

Two, total protein extracts

1, add 10 μ l PMSF (100mM) and 10 μ l Cocktail by 1ml lysate, shake up and be placed on ice that (PMSF will shake It is even to nodeless mesh when can just be mixed with lysate).

2, tissue need to add liquid nitrogen grinding, add lysate (100mg tissue) of the 600 μ l containing PMSF, crack on ice 30min wants frequent waggle to crack cell sufficiently.

3,14000rpm is centrifuged 5min at 4 DEG C after having cracked.(opening centrifuge pre-cooling in advance).

4, the supernatant packing after centrifugation is shifted in clean centrifuge tube and is put in -80 DEG C of preservations.

5, the protein solution for having extracted sample and 5 × sample-loading buffer are boiled into 10min, slow recovery room by 4: 1 mixing Wen Hou is slightly centrifuged, and is put in -20 DEG C of preservations.

Two, protein sample is tentatively quantitative

The kit specification that following operating procedure is provided referring to Development Co., Ltd, Nanjing Keygen Biotech arranges, article No. Are as follows: KGPBCA

1, according to the form below dilution standard sample making standard curve:

Vial	Volume of Diluent	Volume and source of BSA	Final BSA Concentration
				A	70μL H2O	10μL BSA	250μg/ml
B	40μL H2O	40μL A	125μg/ml
				C	45μL H2O	30μL B	50μg/ml
D	40μL H2O	40μL C	25μg/ml
				E	40μL H2O	10μL D	5μg/ml
F	40μL H2O	0	0μg/ml

2, dilution experiment sample: each sample takes 2 μ L, and 38 μ L H2O is added to dilute 20 times.It is arranged in order.

3, it prepares BCA reagent concentration and measures working solution: taking 50 volume A liquid, 1 volume B liquid is added, mixes well, now with existing With.

4, prepare 96 orifice plates, the standard sample and laboratory sample for respectively taking 20 μ L to dilute are in 96 orifice plates.Every hole is added 200 μ L working solution, 37 DEG C are protected from light incubation 30min.

5, light absorption value, that is, OD value, wavelength 560nm are read in microplate reader.

Three, SDS-PAGE electrophoresis

1, before gel electrophoresis, every 1 × electrophoretic buffer of Kong Junyong cleaning.1 × running buffer is added in upper and lower layer electrophoresis tank Liquid, buffer liquid level requires more than loading hole top in the slot of upper layer.

2, according to the loading of sample sequence and loading volume successively loading.

3, electrophoresis: after 80V constant pressure electrophoresis to bromophenol blue to separation gel, 120V constant pressure electrophoresis to the rigid plastic emitting bottom of bromophenol blue Only.

Four, Protein transfer

1, pvdf membrane methanol pre-processes 3~5 seconds, puts to transferring film buffer and infiltrates half an hour.

2, taking-up gel, is put to filter paper, formation gel transfer stack layer, filter paper, gel, pvdf membrane, and filter paper coagulates Glue transfers " sandwich " structure as stack layer.This operation must completely remove bubble.

3, transfer folder is placed by cathode and anode directions.

4, under cryogenic conditions, 100V constant pressure 116 minutes.

Five, immunoblotting analysis

1. take out hybond membrane, TBST rinse 5min, 1 time.

2.5% skimmed milk power solution room temperature is closed 1 hour.

3.TBST washes film 10min, and 1 time.

4. 4 DEG C of suitable primary antibody diluted concentration overnight.

5.TBST washes film 10min, and 4 times.

6. 37 DEG C of incubation 1h of corresponding secondary antibody diluent.

7.TBST washes film 10min, and 4 times.

8. hybond membrane is placed on a transparent plastic sheet, it is careful not to make film dry.

9. chemiluminescence luminous substrate to be equably added to the surface of film with a clean pipettor, and continue reaction 5min。

10. sucking the extra substrate solution of film surface with the filter paper that kit provides, put to magazine.

11. development.

Agent prescription and key instrument, consumptive material, the manufacturer of reagent and article No.

1. 10* electrophoretic buffer

144g Glycine

30g Tirs-Base

10g SDS

It is molten to 1L room temperature preservation after adding 800mL pure water to dissolve.100mL10* buffer adds 900ml pure water to be diluted to when use 1* is used.

2. 10* transferring film buffer

154g Glycine

30g Tris-Base

It is molten to 1L room temperature preservation after adding 800mL pure water to dissolve.200mL first is added in 100mL 10* transferring film buffer when use Alcohol is settled to 1L with pure water again and uses.

③5*Loading Buffer

5mL is dissolved to after adding deionized water dissolving.50 μ L β-sulfydryl second is added in every 1mL Loading Buffer when use Alcohol.

4. Tis buffer formulation (0.5M pH7.6)

Tris-Base 60.57g

After adding 800mL pure water to dissolve, concentrated hydrochloric acid tune pH to 7.6 is settled to 1L.

⑤1*TBS(TBST)

8.5gNaCl first is dissolved with a small amount of pure water, it is rear that 0.5MTis buffer 100mL is added, it is settled to 1L, as 1*TBS. 0.1% Tween-20 is added.

6. RIPA lysate

Be added 100mMPMSF, 100*Cocktail, inhibitors of phosphatases at 1: 100 when use

7. fixing solution

240g sodium thiosulfate

25g anhydrous sodium sulfite

48mL glacial acetic acid

1L is settled to after adding pure water to dissolve.

Four, enzyme-linked immunosorbent assay (ELISA)

The dilution of standard items: according to the form below dilution standard product make standard curve.

200pg/ml	No. 5 standard items	150 μ l standard dilutions are added in the former times of standard items of 150 μ l
			100pg/ml	No. 4 standard items	150 μ l standard dilutions are added in No. 5 standard items of 150 μ l
50pg/ml	No. 3 standard items	150 μ l standard dilutions are added in No. 4 standard items of 150 μ l
			25pg/ml	No. 2 standard items	150 μ l standard dilutions are added in No. 3 standard items of 150 μ l
12.5pg/ml	No. 1 standard items	150 μ l standard dilutions are added in No. 2 standard items of 150 μ l

2. sample-adding: setting blank well (sample and enzyme marking reagent is not added in blank control wells, remaining each step operation is identical), mark respectively Quasi- hole, sample to be tested hole.Standard items are accurately loaded 50 μ l on enzyme mark coating plate, first add sample diluting liquid 40 in sample to be tested hole Then μ l adds 10 μ l of sample to be tested again (the final extension rate of sample is 5 times).Sample is added on ELISA Plate hole bottom by sample-adding, to the greatest extent Amount does not touch hole wall, shakes gently mixing.

3. incubating: using 37 DEG C of incubation 30min of sealing plate film sealing plate postposition.

4. matching liquid: spare after 30 times of concentrated cleaning solutions are diluted 30 times with distilled water

5. washing: carefully taking sealing plate film off, discard liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30s, so It is repeated 5 times, pats dry.

6. enzyme: every hole is added 50 μ l of enzyme marking reagent, except blank well.

7. incubating: operation is the same as 3.

8. washing: operation is the same as 5.

9. colour developing: 50 μ l of color developing agent A is first added in every hole, adds 50 μ l of color developing agent B, and gently concussion mixes, and 37 DEG C are kept away Light colour developing 15min.

10. terminating: every hole adds 50 μ l of terminate liquid, terminates reaction (blue is vertical at this time turns yellow).

11. measurement: with blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance (OD value) in each hole.Measurement should add end Only carried out within 15min after liquid.

The above are merely the preferred embodiment of the present invention, it is noted that for those of ordinary skill in the art, Without departing from the concept of the premise of the invention, various modifications and improvements can be made, these belong to guarantor of the invention Protect range.