CN107102154B - Micro-array chip, preparation method, kit and the detection method of protein expression in a kind of detection cellular stress - Google Patents
Micro-array chip, preparation method, kit and the detection method of protein expression in a kind of detection cellular stress Download PDFInfo
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Abstract
The invention discloses micro-array chip, preparation method, kit and the detection methods of protein expression in a kind of detection cellular stress.On the composition of micro-array chip of the invention, 7 class stress reaction markers have been selected, the variation for the inside expression of cellular proteins that cellular stress is coped under the conditions of different stimulated can be specified, have realized quickly screening and evaluation；The present invention selects the slide of aldehyde radicalization modification as solid carrier, combines specific antibody more stable, activity is higher；The false positive and false negative of spectrum chip testing can be effectively excluded, it is good that the index test high sensitivity of display chip, specificity are tested in cross reaction；Process for preparing microarray chip of the invention is easy, and quickly, whole flow process only needs 2d to complete for detection；High sensitivity；Specificity is good, and cost of manufacture is lower, there is wide industrialization prospect.
Description
Technical field
The present invention relates to chip detection fields, more particularly, to protein expression in a kind of detection cellular stress
Micro-array chip, preparation method, kit and detection method.
Background technique
Cellular stress is protokaryon or eukaryotic by various apparent environmental changes or meets with ray, and active oxygen etc. causes
When macromolecular is damaged, a series of adaptive changes of generation eventually lead to the change of gene expression, enhance cell antibody Monoclonal ability
Survival ability in adverse conditions.Currently, common cellular stress process mainly has Apoptosis, cell autophagy, cell
Proliferation, cell migration, cell stemness, protein translation etc..
Different cellular stress can be generated for different environmental stimulis, and it is poor to be mainly reflected in intracellular protein expression
On different.Such as Nature enemy is carried out to cell, for cell in order to cope with no nutrition intake situation, interior substrate protein can be double by ribosomes
Tunic package, then the outer membrane of autophagy vesicle is merged with lysosome membrane or tonoplast, releases the blister of package substrate protein
Structure is finally degraded into lysosome or vacuole.In this course, the albumen such as LC3 and p62 can occur significantly to drop
Solution, this also becomes the significant albumen of cell autophagy generation.
In current scientific research, corresponding one to two cellular stress mistake is generally only focused on for different research directions
Journey, lack to environmental stimuli cellular stress comprehensively, accurately and rapidly detection means.Under normal circumstances, carry out molecular mechanism and thin
Extracellular molecule biology still generallys use the routine proteins such as WB, Elisa, immunohistochemistry expression means of testing, but these means
Test process is cumbersome, technology is complicated, expensive reagents.
In order to aiming at the problem that albumen carries out qualitative or quantitative analysis under the conditions of cellular stress with high throughput, protein chip
Exploitation it is very necessary.However the exploitation of protein chip has the difficult point for much needing to overcome: 1, the basis of protein chip is anti-
Original antibody compatible reaction, when antibody is fixed on carrier surface, specificity and affinity are restricted, and specificity often occur
It is low, in conjunction with unstable situation；2, abundance of the different protein in clinical sample is different, the affinity of antibody antigen reaction
Difference, sample dilution are improper, it is easy to the case where false negative occur；3, there is cross reactions between protein, seriously affect
The accuracy of testing result needs to overcome the difficulty of cross reaction bigger with the increase of detection antibody levels.Only overcome with
Upper difficult point, it is specific good just to prepare, and cross reaction is small, the high protein chip of accuracy rate, to avoid false positive and vacation yin
The appearance of property.
Summary of the invention
It is an object of the present invention to provide a kind of micro-array chips of protein expression in detection cellular stress；
Another object of the present invention is to provide the micro-array chips of protein expression in a kind of detection cellular stress
Preparation method；
Another object of the present invention is to provide a kind of kits of protein expression in detection cellular stress；
Another object of the present invention is to provide a kind of detection methods of protein expression in cellular stress；
The technical solution used in the present invention is:
The micro-array chip of protein expression in a kind of detection cellular stress comprising solid carrier and array type are fixed
The independently specific antibody of stress reaction marker on a solid carrier；The stress reaction marker is following all kinds of
One kind or multiclass of marker protein: cell Proliferation marker protein, Apoptosis marker protein, cell autophagy marker protein, cell
Migrate marker protein, protein translation marker protein, cell stemness marker protein, cellular stress marker protein.
As the preferred of above-mentioned micro-array chip, stress reaction marker is one kind or more of following all kinds of marker proteins
Class, all kinds of marker proteins contain multiple independently marker proteins, in which:
Cell Proliferation marker protein is PCNA, Ki67, CyclinD1；
Apoptosis marker protein be PRAP, Caspase3, Caspase9, Caspase8, Caspase12, Bcl-2,
Bad；
Cell autophagy marker protein is LC3- I, LC3- II, p62, Atg3, Atg5, Atg7, Atg12；
Cell migration marker protein is FAK, cofilin, profilin, CDC42, α-Tubulin；
Protein translation marker protein is eIF2B, eIF4EBP1, p70S6K, mTOR, eIF4E, S6RP；
Cell stemness marker protein be Oct3/4, SSEA-3/4/1, SOX2, Nanog, CD34, ABCG2, STRO-1,
Nestin,PSA-NCAM,Sca-1,CD44,CD166,CD133；
Cellular stress marker protein is HIF1, HIF2, P53, ATF4, HSP70, HSP90, ROS, Nrf2, ATF6.
As the preferred of above-mentioned micro-array chip, solid carrier is the slide of aldehyde radicalization modification.
As the preferred of above-mentioned micro-array chip, array type is gone back on the solid carrier of micro-array chip and is fixed with positive control
The specific antibody of albumen and/or the specific antibody of negative control protein.
A method of the micro-array chip preparing protein expression in detection cellular stress includes the following steps:
It is 300~400 μm by distance between point, point sample amount is 350~450pL, is the special of 300~500 μ g/mL by concentration
Property antibody-solutions with array type point sample on the slide that aldehyde radicalization is modified, fixed after drying at room temperature, closing after incubation, obtains micro-
Array chip.
As the preferred of the above method, the method for aldehyde radicalization modification is: the slide cleaned up is used containing 4.0~6.0%
(V/V) ethanol solution of aminomethoxy silane handles 20~40min, dry, then with containing 1.5~3.5% (V/V) glutaraldehydes
PBS solution handles 50~70min, dry, obtains the slide of aldehyde radicalization modification.
The kit of the micro-array chip of protein expression, contains above-mentioned microarray core in a kind of detection cellular stress
Piece.
As the preferred of mentioned reagent box, also contain cell pyrolysis liquid, Tissue lysates, dilution, washing lotion, enzyme mark two
One of anti-, luminous substrate is a variety of.
As the preferred of mentioned reagent box, the dilution is protein stabiliser；
And/or washing lotion is neutral buffered liquid；
And/or ELIAS secondary antibody is the secondary antibody of horseradish peroxidase-labeled；
And/or luminous substrate is fluorescein 555.
The detection method of protein expression, includes the following steps: in cellular stress
Cell sample to be checked is cracked, protein lysate is obtained, point sample is described in any item in Claims 1 to 4
It on micro-array chip, is incubated for, cleaning；ELIAS secondary antibody is added, is incubated for, cleaning；Luminous substrate is added, is incubated for, cleaning；It reads
Fluorescence signal is normalized using positive control albumen as internal reference by standard of internal reference, obtains half-quantitative detection result.
The beneficial effects of the present invention are:
(1) on the composition of micro-array chip of the invention, 7 classes has been selected to amount to 50 stress reaction markers, it can be simultaneously
Detection include cell Proliferation, Apoptosis, cell migration, cell autophagy, protein translation, cell stemness, cellular stress these
The protein expression of aspect can specify the change for the inside expression of cellular proteins that cellular stress is coped under the conditions of different stimulated
Change, to realize quickly screening and evaluation, provides more direction selections for numerous scientific research personnel；
(2) micro-array chip of the invention selects the slide of aldehyde radicalization modification as solid carrier, using aminomethoxy
Silane and glutaraldehyde successively carry out amination and aldehyde radicalization modification, form the aldehyde radical of activation in surface of glass slide, the ammonia with protein
Base condensation reaction forms covalent bond, combines specific antibody more stable, activity is higher.
(3) present invention by microarray point sample design, point sample concentration it is preferred, two anti-luminous substrate polymolecular systems
The optimization such as building, is prepared into the micro-array chip that index is more, flux is high, rate is fast, 300 samples of a performance detection, and point
Sample amount is low, it is only necessary to 100 μ L, and the false positive and false negative of spectrum chip testing can be effectively excluded, improve the accurate of detection
Degree finds that accuracy rate is greater than 82% by great amount of samples analysis；Overcome the detection such as WesternBlot, Elisa, immunohistochemistry
The single drawback of index, furthermore the index test high sensitivity, specificity of cross reaction test display chip are good；
(4) process for preparing microarray chip of the invention is easy, and quickly, whole flow process only needs complete for 2 days, and makes for detection
Cost is relatively low for work, there is wide industrialization prospect.
(5) surface modification of the invention by slide, improvement, are prepared a kind of novel chip, can greatly improve
The adsorption capacity and affinity of albumen index.
Detailed description of the invention
Fig. 1: lung cell A549 is to apoptosis marker protein expression quantity normalized under cis-platinum incentive condition as a result, figure
In, A:WesternBlot test result；B: the test result of micro-array chip of the present invention；
Fig. 2: autophagy marker protein expression quantity normalized under the conditions of liver cancer cells Hep G2 Nature enemy is as a result, figure
In, A:WesternBlot test result；B: the test result of micro-array chip of the present invention.
Specific embodiment
Following embodiment is not limited to the scope of the invention to illustrate the present invention.Without departing substantially from spirit of that invention and
Under real situation, modifies or replaces, all belong to the scope of the present invention to what method of the invention, step, condition were done.
The formula of reagent of the present invention is as follows:
PBS solution: 3.58g Na2HPO4、0.25g NaH2PO4, 8g NaCl, 0.2g KCl, 1L deionized water；
Confining liquid: 0.1g BSA, 100mL PBS；
1%PBST solution: 5mL Tween20,500mL PBS；
5%BSA solution: 5g BSA, 100mL PBS.
Fluorescein 555 is purchased from Streptavidin, HiLyte FluorTM555 conjugated
The goat anti-human igg of horseradish peroxidase-labeled is purchased from Jackson ImmunoResearch
The preparation method of the micro-array chip of protein expression in embodiment 1, detection cellular stress
(1) preparation of the slide of aldehyde radicalization modification
95 DEG C are heated to deionized water 800mL, about washing powder 5g is added, new slide is immersed in 3h in washing powder water；
Slide is taken out, impregnates 1h in the KOH deionized water solution of 1mM；Slide is taken out, is cleaned 3 times, each 5min with deionized water,
It dries, the slide cleaned up；
By alcohol solution dipping 30min of the slide cleaned up containing 5% aminomethoxy silane (V/V)；Take out glass
Piece is washed 3 times, each 5min with acetone；It is cleaned 3 times with deionized water again, each 5min is dried with nitrogen, and obtains amination modification
Slide；
The PBS solution containing 2.5% glutaraldehyde (V/V) is used to impregnate 1h the slide of amination modification；Slide is taken out, PBS is used
It washes 3 times, each 5min；It is cleaned 3 times with deionized water again, each 5min is dried with nitrogen, and obtains the slide of aldehyde radicalization modification.
(2) the point print of chip
By the spy of stress reaction marker independently specific antibody (Sigma-Aldrich, USA) and positive control albumen
The specific antibody of heterogenetic antibody and negative control protein is diluted to 400 μ g/mL with PBS respectively, draws each 30 μ of protein sample
L is added in 384 orifice plates, wherein three anodes are respectively: 200 times of diluted fluoresceins, 555,400 times of diluted fluoresceins
555,800 times of diluted fluoresceins 555, cathode PBS.With will be independently special with point sample instrument (Nano-PlottertmNP 2.1)
On the slide that heterogenetic antibody point sample is modified to ready aldehyde radicalization, point sample amount is 400pL, and distance is 400 μm between point, matrix feelings
Condition is as shown in table 1, and after the completion of printing operation, slide is placed in drying at room temperature.
Above-mentioned stress reaction marker is following 7 class marker proteins, in which:
Cell Proliferation marker protein is PCNA, Ki67, CyclinD1；
Apoptosis marker protein be PRAP, Caspase 3, Caspase 9, Caspase 8, Caspase12, Bcl-2,
Bad；
Cell autophagy marker protein is LC3- I, LC3- II, p62, Atg3, Atg5, Atg7, Atg12；
Cell migration marker protein is FAK, cofilin, profilin, CDC42, α-Tubulin；
Protein translation marker protein is eIF2B, eIF4EBP1, p70S6K, mTOR, eIF4E, S6RP；
Cell stemness marker protein be Oct3/4, SSEA-3/4/1, SOX2, Nanog, CD34, ABCG2, STRO-1,
Nestin,PSA-NCAM,Sca-1,CD44,CD166,CD133；
Cellular stress marker protein is HIF1, HIF2, P53, ATF4, HSP70, HSP90, ROS, Nrf2, ATF6.
Positive control albumen is GADPH, beta-actin；
Negative control protein is BSA, Blue, biotin-protein；
Every chip has anode and cathode to compare.
Table 1, microarray chip point sample array are distributed schematic table
| Positive 1 | Casp3 | P62 | CDC42 | Positive 8 | STRO-1 | CD133 | Nrf2 |
| Positive 2 | Caps9 | Atg3 | α-Tubulin | Oct3/4 | Positive 11 | Positive 14 | ATF6 |
| Positive 3 | Caps8 | Atg5 | Positive 7 | SSEA-3/4/1 | Nestin | HIF1 | Positive 15 |
| Negative | Casp12 | Atg7 | eIF2B | SOX2 | Positive 12 | HIF2 | |
| PCNA | Bcl-2 | Atg12 | eIF4EBP1 | Nanog | PSA-NCAM | P53 | |
| Ki67 | Bad | Positive 6 | p70S6K | Positive 9 | Sca-1 | ATF4 | |
| CyclinD1 | Positive 5 | FAK | mTOR | CD34 | Positive 13 | HSP70 | |
| Positive 4 | LC3-1 | cofilin | eIF4E | Positive 10 | CD44 | HSP90 | |
| PARP | LC3-2 | profilin | S6RP | ABCG2 | CD166 | ROS |
(3) post-processing of micro-array chip
Dried slide is fixed with 16 hole slide incubators, 100 μ L confining liquids are added in every hole, are incubated for closing 30min；
After the completion of closing, confining liquid is removed, is dried, that is, completes the preparation of micro-array chip.
The preparation of the kit of protein expression in embodiment 2, detection cellular stress
The kit of protein expression in a kind of detection cellular stress, including following component: (1) it is anti-to detect cellular stress
Answer micro-array chip, (2) cell pyrolysis liquid, (3) Tissue lysates, (4) dilution, (5) washing lotion, (6) enzyme of middle protein expression
Mark secondary antibody, (7) luminous substrate；The selection and effect of each component are as follows:
(1) micro-array chip of protein expression in cellular stress: the preferably micro-array chip of embodiment 1 is detected；
(2) cell pyrolysis liquid: containing Loading buffer, protease inhibitors is purchased from the green skies biotechnology in Beijing
Co., Ltd；
(3) Tissue lysates are purchased from the green limited public affairs of skies biotechnology in Beijing containing Loading buffer and pmsf
Department；
(4) dilution: being protein stabiliser, and the present embodiment is 5%BSA solution；Effect is that dilution testing protein can extremely be examined
Survey concentration range；
(5) washing lotion: being neutral buffered liquid, and the present embodiment is 1%PBST solution, and effect is to be incubated for egg in solid carrier surface
After white sample and ELIAS secondary antibody, solid carrier surface unbonded antibody and ELIAS secondary antibody need to be washed with washing lotion；
(6) ELIAS secondary antibody: the secondary antibody of horseradish peroxidase-labeled, the present embodiment are the sheep of horseradish peroxidase-labeled
Anti-human igg；Effect is that stress protein in cell protein can be with an anti-binding with the anti-binding on solid carrier, secondary antibody；
(7) luminous substrate: the present embodiment is fluorescein 555, and effect is that the marker on secondary antibody can be anti-with luminous substrate
It answers, to issue detectable light；
Mentioned reagent box can quickly and easily detect cell Proliferation, apoptosis, autophagy, proliferation, migration, protein translation,
Stemness, stress correlating markings protein expression the case where, by great amount of samples analysis find accuracy rate be greater than 82%.
The detection method of protein expression in embodiment 3, cellular stress
The detection method of protein expression, includes the following steps: in cellular stress
(1) Tissue lysates and/or cell pyrolysis liquid are utilized, will be cracked to sample, protein lysate is obtained, use is dilute
It releases liquid and is diluted to suitable detectable concentration；
(2) the 100 diluted protein lysates of μ L are added in every hole on micro-array chip, and room temperature is incubated for 4h, removes unreacted
Protein lysate sample is respectively washed 5 times with washing lotion and PBS solution, drying；
(3) 100 μ L, the goat-anti people of 1000 times of diluted horseradish peroxidase-labeleds is added in every hole on micro-array chip
IgG, room temperature are incubated for 4h, remove unreacted ELIAS secondary antibody solution, be respectively washed 5 times with washing lotion, PBS, dry；
(4) 1000 times of diluted fluoresceins 555 of 100 μ L are added in every hole on micro-array chip, are protected from light and are incubated for 1h.Remove
Extra fluorescein 555 is respectively washed 5 times with washing lotion, PBS, drying；
(5) slide is loaded into scanner (LuxScanTM10K-B), sweep parameter: Power=100%, PMT is set
=550, read fluorescence signal.
(6) it using positive control albumen as internal reference, is normalized as unit of internal reference, obtains half-quantitative detection knot
Fruit.
Proliferation and apoptosis, autophagy marker protein index cross reaction test in embodiment 4, system of the present invention
Since the albumen that cross reaction is related to is relatively more, only show that representative proliferation and apoptosis, autophagy proteins are anti-
The cross reaction test result of body, test method are as follows: test of the antibody to the cross reaction of time.Different antibodies chip is first
It is incubated for and cultivates with single antigen (concentration 150ng/mL), develop a film according to the standard of developing a film of embodiment 3.It is corresponding with every kind of antigen
Detection antibody response, be incubated for through Cy3- Streptavidin, read after chip scanning.It is cross with the capture antibody of every kind of antigen
Axis is drawn so that antigen and corresponding detection antibody is added as the longitudinal axis.
The result of table 2, proliferation and apoptosis marker protein antibody cross reaction
| Antibody/antigen | PCNA | Ki67 | CyclinD1 | PARP | Casp3 | Caps9 | Caps8 | Casp12 | Bcl-2 | Bad |
| PCNA | 34512 | 123 | 132 | 142 | 99 | 92 | 93 | 153 | 241 | 215 |
| Ki67 | 214 | 28754 | 270 | 345 | 298 | 330 | 321 | 361 | 353 | 321 |
| CyclinD1 | 192 | 201 | 38134 | 230 | 258 | 294 | 201 | 249 | 251 | 301 |
| PARP | 183 | 102 | 294 | 23415 | 284 | 222 | 304 | 320 | 341 | 329 |
| Casp3 | 201 | 153 | 271 | 119 | 31289 | 348 | 602 | 503 | 559 | 542 |
| Caps9 | 198 | 183 | 263 | 140 | 132 | 34129 | 421 | 321 | 298 | 342 |
| Caps8 | 281 | 152 | 231 | 139 | 162 | 219 | 41093 | 320 | 184 | 156 |
| Casp12 | 241 | 182 | 221 | 156 | 162 | 239 | 302 | 29120 | 223 | 234 |
| Bcl-2 | 199 | 153 | 290 | 172 | 135 | 212 | 310 | 310 | 32194 | 169 |
| Bad | 210 | 132 | 301 | 130 | 142 | 251 | 224 | 301 | 341 | 30123 |
The result of table 3, autophagy marker protein antibody cross reaction
| Antibody/antigen | LC3-1 | LC3-2 | P62 | CDC42 | Atg3 | Atg5 | Atg7 | Atg7 |
| LC3-1 | 29021 | 313 | 430 | 394 | 403 | 443 | 242 | 209 |
| LC3-2 | 430 | 30120 | 351 | 372 | 443 | 393 | 402 | 432 |
| P62 | 392 | 390 | 37120 | 352 | 282 | 342 | 309 | 302 |
| CDC42 | 320 | 331 | 321 | 32102 | 269 | 376 | 234 | 332 |
| Atg3 | 424 | 342 | 352 | 452 | 34021 | 403 | 441 | 345 |
| Atg5 | 392 | 402 | 362 | 442 | 293 | 29210 | 362 | 346 |
| Atg7 | 340 | 453 | 423 | 456 | 235 | 345 | 40212 | 432 |
| Atg12 | 321 | 342 | 342 | 463 | 309 | 285 | 349 | 38921 |
Statistical result as shown in table 2 and table 3, every kind of antibody to can specifically identify oneself detection antigen and and other
Antigen there is no cross reaction.
The detection of apoptosis marker protein under embodiment 5, cis-platinum incentive condition
Lung cancer using the detection method and WesternBlot detection method of embodiment 3, after 1.5 μM of cis-platinums to be stimulated to 12h
A549 cell sample is detected, and is control with the lung cancer A549 cell sample not stimulated.Stress protein chip results data
With analysis 6.0 software of software Gene Pro carries out fluorescence signal reading, reading obtain each index before and after the processing
Fluorescence signal value is normalized using positive control Protein G APDH as internal reference by standard of internal reference, soft with Grapa6.0
Part carries out interpretation of result (t inspection).
As a result signal is as shown in Figure 1, A is the method for the present invention testing result in figure, and B is WesternBlot detection knot in figure
Fruit does not show in figure since the expression of Bcl-12 index is lower in the cell sample, compared with the control group not stimulated, two kinds
Detection method show apoptosis pathway marker protein PARP lower, and marker protein Casp3, Casp8, Casp9, Caspase12,
Bad is then raised, and the method for the present invention and WesternBlot testing result difference are little, can accurately react stress reaction
The variation of albumen.
The detection of autophagy marker protein under the conditions of embodiment 6, Nature enemy
Using the detection method and WesternBlot detection method of embodiment 3, by the liver cancer Hep after Nature enemy 12h
G2 cell sample is detected, and is control with the liver cancer Hep G2 cell sample of non-Nature enemy.Stress protein chip results
Data carry out fluorescence signal reading with analysis 6.0 software of software Gene Pro, before reading obtains the processing of each index
Fluorescence signal value afterwards is normalized using internal reference GAPDH as standard, and carrying out interpretation of result with Grapa6.0 software, (t is examined
It tests).
As a result signal is as shown in Fig. 2, A is the method for the present invention testing result in figure, and B is WesternBlot detection knot in figure
Fruit does not show in figure since remaining 4 index expression is lower in the cell sample, compared with the control group not stimulated, two kinds
Detection method shows that cell autophagy marker protein LC3-1, p62P are lowered, and marker protein LC3- II is then raised, and this hair
Bright method and WesternBlot testing result difference are little, can accurately react the variation of stress response protein.
Claims (8)
1. the micro-array chip of protein expression in a kind of detection cellular stress comprising solid carrier and array type are fixed on
The independently specific antibody of stress reaction marker on solid carrier；
The stress reaction marker is following a variety of marker proteins, is independently indicated containing multiple again in every kind of marker protein
Albumen, in which:
Cell Proliferation marker protein is PCNA, Ki67, CyclinD1；
Apoptosis marker protein is PRAP, Caspase3, Caspase9, Caspase8, Caspase12, Bcl-2, Bad；
Cell autophagy marker protein is LC3- I, LC3- II, p62, Atg3, Atg5, Atg7, Atg12；
Cell migration marker protein is FAK, cofilin, profilin, CDC42, α-Tubulin；
Protein translation marker protein is eIF2B, eIF4EBP1, p70S6K, mTOR, eIF4E, S6RP；
Cell stemness marker protein be Oct3/4, SSEA-3/4/1, SOX2, Nanog, CD34, ABCG2, STRO-1, Nestin,
PSA-NCAM,Sca-1,CD44,CD166,CD133；
Cellular stress marker protein is HIF1, HIF2, P53, ATF4, HSP70, HSP90, ROS, Nrf2, ATF6；
The solid carrier is the slide of aldehyde radicalization modification.
2. micro-array chip according to claim 1, it is characterised in that: go back array type on the solid carrier of micro-array chip
It is fixed with the specific antibody of positive control albumen and/or the specific antibody of negative control protein.
3. a kind of prepare the microarray core such as protein expression in the described in any item detection cellular stress of claim 1~2
The method of piece, includes the following steps:
It is 300~400 μm by distance between point, point sample amount is 350~450pL, and the specificity that concentration is 300~500 μ g/mL is resisted
Liquid solution is with array type point sample on the slide that aldehyde radicalization is modified, and fixed after drying at room temperature, closing after incubation, obtains microarray
Chip.
4. according to the method described in claim 3, it is characterized by: the method for aldehyde radicalization modification is: the slide that will be cleaned up
20~40min is handled with the ethanol solution of the silane Han 4.0~6.0%V/V aminomethoxy, it is dry, then with containing 1.5~3.5%
The PBS solution of V/V glutaraldehyde handles 50~70min, dry, obtains the slide of aldehyde radicalization modification.
5. the kit of the micro-array chip of protein expression in a kind of detection cellular stress, it is characterised in that: containing having the right
It is required that 1~2 described in any item micro-array chips.
6. kit according to claim 5, it is characterised in that: also contain cell pyrolysis liquid, Tissue lysates, dilution
One of liquid, washing lotion, ELIAS secondary antibody, luminous substrate are a variety of.
7. kit according to claim 6, it is characterised in that: the dilution is protein stabiliser；
And/or washing lotion is neutral buffered liquid；
And/or ELIAS secondary antibody is the secondary antibody of horseradish peroxidase-labeled；
And/or luminous substrate is fluorescein 555.
8. the detection method of protein expression in cellular stress includes the following steps: to crack cell sample to be checked, obtain
Protein lysate is obtained, point sample is incubated on the described in any item micro-array chips of claim 1~2, cleaning；Add enzyme mark
Secondary antibody is incubated for, cleaning；Luminous substrate is added, is incubated for, cleaning；Fluorescence signal is read, using positive control albumen as internal reference, with
Internal reference is normalized for standard, obtains half-quantitative detection result.
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