CN107102154A - 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 a kind of micro-array chip, preparation method, kit and detection method for detecting protein expression in cellular stress.On the composition of the micro-array chip of the present invention, 7 class stress reaction marks have been selected, the change of the inside expression of cellular proteins that cellular stress is tackled under the conditions of different stimulated can be specified, have realized quick screening with evaluating；The present invention selects the slide that aldehyde radicalization is modified 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, the index test sensitivity of cross reaction test display chip is high, specific good；The process for preparing microarray chip of the present invention is easy, and detection is quick, and whole flow process only needs 2d to complete；Sensitivity is high；Specificity is good, and cost of manufacture is relatively low, there is wide industrialization prospect.
Description
Technical field
The present invention relates to chip detection field, protein expression in cellular stress is detected more particularly, to a kind of
Micro-array chip, preparation method, kit and detection method.
Background technology
Cellular stress is protokaryon or eukaryotic by various obvious environmental changes or meets with ray, and active oxygen etc. causes
When macromolecular is damaged, a series of adaptations of generation ultimately result in the change of gene expression, strengthen cell antibody Monoclonal ability
Survival ability in adverse conditions.At present, common cellular stress process mainly has Apoptosis, cell autophagy, cell
Propagation, cell migration, cell dryness, protein translation etc..
Different cellular stress can be produced for different environmental stimulis, intracellular protein expression are mainly reflected in poor
On different.Nature enemy is for example carried out to cell, cell is in order to tackle no nutrition intake situation, and interior substrate protein can be double by ribosomes
Tunic is wrapped up, and then the outer membrane of autophagy vesicle is merged with lysosome membrane or tonoplast, discharges the blister of parcel substrate protein
Structure is into lysosome or vacuole, and most it is degraded at last.In this course, obvious drop can occur for the albumen such as LC3 and p62
Solution, this also turns into the significant albumen that cell autophagy occurs.
In current scientific research, corresponding one to two cellular stress mistake is typically only focused on for different research directions
Journey, lack stimulate to external world cellular stress comprehensively, accurately and rapidly detection means.Generally, molecular mechanism is carried out and thin
Extracellular molecule biology is still generally using WB, Elisa, the routine protein such as SABC expression means of testing, but these means
Test process is cumbersome, technical sophistication, expensive reagents.
The problem of in order to carry out qualitative or quantitative analysis for albumen under the conditions of cellular stress with high throughput, protein chip
Exploitation it is very necessary.But there is the difficult point for much needing to overcome in the exploitation of protein chip：1st, the basis of protein chip is anti-
Original antibody compatible reaction, when antibody is fixed on carrier surface, its specificity and affinity are restricted, and specificity often occur
It is low, with reference to unstable situation；2nd, abundance of the different protein in clinical sample is different, the affinity of antibody antigen reaction
Difference, sample dilution is improper, it is easy to the situation of false negative occur；3rd, cross reaction is there is between protein, had a strong impact on
The degree of accuracy of testing result, as the increase of detection antibody levels is, it is necessary to overcome the difficulty of cross reaction bigger.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, so as to avoid false positive and vacation cloudy
The appearance of property.
The content of the invention
It is an object of the present invention to provide a kind of micro-array chip for detecting protein expression in cellular stress；
Another object of the present invention is to provide the micro-array chip of protein expression in a kind of detection cellular stress
Preparation method；
Another object of the present invention is to provide a kind of kit for detecting protein expression in cellular stress；
Another object of the present invention is to provide a kind of detection method 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, it includes solid carrier and array type is fixed
The independently specific antibody of stress reaction mark on a solid support；Described stress reaction mark is following all kinds of
A class or multiclass for marker protein：Cell propagation marker protein, Apoptosis marker protein, cell autophagy marker protein, cell
Migrate marker protein, protein translation marker protein, cell dryness marker protein, cellular stress marker protein.
As the preferred of above-mentioned micro-array chip, stress reaction mark is a class or many for following all kinds of marker proteins
Class, all kinds of marker proteins contain multiple independently marker proteins, wherein：
Cell propagation 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 dryness 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 that aldehyde radicalization is modified.
Array type, which is gone back, as the preferred of above-mentioned micro-array chip, on the solid carrier of micro-array chip is fixed with positive control
The specific antibody of albumen and/or the specific antibody of negative control protein.
A kind of method for preparing the micro-array chip of protein expression in detection cellular stress, comprises 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, is obtained micro-
Array chip.
As the preferred of the above method, the method for aldehyde radicalization modification is：The slide cleaned up is used and contains 4.0~6.0%
(V/V) ethanol solution of aminomethoxy silane handles 20~40min, dries, then with containing 1.5~3.5% (V/V) glutaraldehydes
PBS solution handles 50~70min, dries, and obtains the slide of aldehyde radicalization modification.
The kit of the micro-array chip of protein expression in a kind of detection cellular stress, contains above-mentioned microarray core
Piece.
As the preferred of mentioned reagent box, also containing cell pyrolysis liquid, Tissue lysates, dilution, washing lotion, enzyme mark two
One or more in anti-, luminous substrate.
As the preferred of mentioned reagent box, the dilution is protein stabiliser；
And/or washing lotion is neutral buffered liquid；
And/or the secondary antibody that ELIAS secondary antibody is horseradish peroxidase-labeled；
And/or luminous substrate is fluorescein 555.
The detection method of protein expression, comprises the following steps in cellular stress：
Cell sample to be checked is cracked, protein lysate is obtained, point sample is in described in any one of Claims 1 to 4
On micro-array chip, it is incubated, cleaning；ELIAS secondary antibody is added, is incubated, cleaning；Luminous substrate is added, is incubated, cleaning；Read
Fluorescence signal, using positive control albumen as internal reference, is normalized by standard of internal reference, obtains half-quantitative detection result.
The beneficial effects of the invention are as follows：
(1) on the composition of micro-array chip of the invention, 7 classes have been selected to amount to 50 stress reaction marks, can be simultaneously
Detection include cell propagation, Apoptosis, cell migration, cell autophagy, protein translation, cell dryness, cellular stress these
The protein expression of aspect, can specify the change of the inside expression of cellular proteins that cellular stress is tackled under the conditions of different stimulated
Change, so as to realize quick screening and evaluate, more set directions are provided for numerous scientific research personnel；
(2) micro-array chip of the invention selects the slide that aldehyde radicalization is modified as solid carrier, using aminomethoxy
Silane and glutaraldehyde successively carry out amination and aldehyde radicalization modification, in the aldehyde radical of surface of glass slide formation activation, the ammonia with protein
Base condensation reaction, forms covalent bond, combines specific antibody more stable, activity is higher.
(3) present invention is by the design of microarray point sample, point sample concentration it is preferred, two anti-luminous substrate polymolecular systems
The optimization such as structure, is prepared into the micro-array chip that index is more, flux is high, speed is fast, 300 samples of a performance detection, and point
Sample amount is low, it is only necessary to 100 μ L, and can effectively exclude the false positive and false negative of spectrum chip testing, improves the accurate of detection
Degree, finds that accuracy rate is more than 82% by great amount of samples analysis；Overcome the detection such as WesternBlot, Elisa, SABC
The drawbacks of index is single, the index test sensitivity of cross reaction test display chip is high, specific good in addition；
(4) process for preparing microarray chip of the invention is easy, and detection is quick, and whole flow process only needs complete for 2 days, and makes
Make that cost is relatively low, there is wide industrialization prospect.
(5) present invention by the surface modification of slide, improve, prepare a kind of new chip, can greatly improve
The adsorption capacity and affinity of albumen index.
Brief description of the drawings
Fig. 1：Lung cell A549 is to apoptosis marker protein expression quantity normalized result under cis-platinum incentive condition, figure
In, A：WesternBlot test results；B：The test result of micro-array chip of the present invention；
Fig. 2：Autophagy marker protein expression quantity normalized result under the conditions of liver cancer cells Hep G2 Nature enemies, figure
In, A：WesternBlot test results；B：The test result of micro-array chip of the present invention.
Embodiment
Following examples are not limited to the scope of the invention to illustrate the present invention.Without departing substantially from spirit of the invention and
Under real situation, modification or replacement that method, step, condition to the present invention are made belong to the scope of the present invention.
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 waters；
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, 1h is soaked with 1mM KOH deionized water solutions；Slide is taken out, is cleaned with deionized water 3 times, each 5min,
Dry, 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；Cleaned again with deionized water 3 times, each 5min, nitrogen drying obtains amination modification
Slide；
PBS solution immersion 1h of the slide that amination is modified containing 2.5% glutaraldehyde (V/V)；Slide is taken out, PBS is used
Wash 3 times, each 5min；Cleaned again with deionized water 3 times, each 5min, nitrogen drying obtains the slide of aldehyde radicalization modification.
(2) the point print of chip
By the spy of stress reaction mark 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 μ of protein sample 30
L is added in 384 orifice plates, wherein, three positive poles are respectively：200 times dilution fluorescein 555,400 times dilution fluorescein
555th, the fluorescein 555 of 800 times of dilutions, negative pole is 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 that distance is 400 μm, matrix feelings between 400pL, point
Slide as shown in table 1, after the completion of printing operation, is placed in drying at room temperature by condition.
Above-mentioned stress reaction mark is following 7 class marker proteins, wherein：
Cell propagation 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 dryness 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 distribution signal 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 per hole, closing 30min is incubated；
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) detection cellular stress is anti-
Micro-array chip, (2) cell pyrolysis liquid, (3) Tissue lysates, (4) dilution, (5) washing lotion, (6) enzyme of protein expression in answering
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 is detected：The preferably micro-array chip of embodiment 1；
(2) cell pyrolysis liquid：Containing Loading buffer, protease inhibitors is purchased from the green skies biotechnology in Beijing
Co., Ltd；
(3) Tissue lysates, containing Loading buffer and pmsf, are purchased from the green limited public affairs of skies biotechnology in Beijing
Department；
(4) dilution：It is protein stabiliser, the present embodiment is 5%BSA solution；Effect is that dilution testing protein can extremely be examined
Survey concentration range；
(5) washing lotion：It is neutral buffered liquid, the present embodiment is 1%PBST solution, and effect is to be incubated egg in solid carrier surface
After white sample and ELIAS secondary antibody, solid carrier surface uncombined 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 is 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 its effect is that the label on secondary antibody can be anti-with luminous substrate
Should, so as to send detectable light；
Mentioned reagent box can quickly and easily detect cell propagation, apoptosis, autophagy, propagation, migration, protein translation,
Dryness, stress correlating markings protein expression situation, by great amount of samples analysis find accuracy rate be more than 82%.
The detection method of protein expression in embodiment 3, cellular stress
The detection method of protein expression, comprises the following steps in cellular stress：
(1) Tissue lysates and/or cell pyrolysis liquid are utilized, will treat that sample is cracked, acquisition protein lysate, with dilute
Release liquid and be diluted to suitable detectable concentration；
(2) protein lysate of 100 μ L dilutions is added per hole on micro-array chip, normal temperature is incubated 4h, removes unreacted
Protein lysate sample, is respectively washed 5 times with washing lotion and PBS solution, is dried；
(3) 100 μ L, the goat-anti people of the horseradish peroxidase-labeled of 1000 times of dilutions are added per hole on micro-array chip
IgG, normal temperature is incubated 4h, removes unreacted ELIAS secondary antibody solution, is respectively washed with washing lotion, PBS 5 times, dries；
(4) fluorescein 555 of 100 μ L 1000 times of dilutions is added per hole on micro-array chip, lucifuge is incubated 1h.Remove
Unnecessary fluorescein 555, is respectively washed 5 times with washing lotion, PBS, is dried；
(5) slide is loaded into scanner (LuxScanTM10K-B), sweep parameter is set：Power=100%, PMT
=550, read fluorescence signal.
(6) using positive control albumen as internal reference, it is normalized in units of internal reference, obtains half-quantitative detection knot
Really.
Propagation and apoptosis, autophagy marker protein index cross reaction test in embodiment 4, system of the present invention
Because the albumen that cross reaction is related to is relatively more, only show that representative propagation and apoptosis, autophagy proteins are anti-
The cross reaction test result of body, method of testing is as follows：Test of the antibody to the cross reaction of time.Different antibodies chip is first
It is incubated and cultivates with single antigen (concentration 150ng/mL), is developed 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 through Cy3- Streptavidins, reading after chip scanning.Using the capture antibody of every kind of antigen as horizontal stroke
Axle, is drawn using adding antigen and correspondence detection antibody as the longitudinal axis.
The result of table 2, propagation 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 recognize 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
Using the detection method and WesternBlot detection methods of embodiment 3,1.5 μM of cis-platinums are stimulated into the lung cancer after 12h
A549 cell samples are detected, using the lung cancer A549 cell sample that does not stimulate as control.Stress protein chip results data
Carry out fluorescence signal reading with the software of analysis software Gene Pro 6.0, read obtain each index before and after the processing
Fluorescence signal value, using positive control Protein G APDH as internal reference, is normalized by standard of internal reference, soft with Grapa6.0
Part carries out interpretation of result (t inspections).
As a result signal is as shown in figure 1, A is the inventive method testing result in figure, and B is that WesternBlot detects knot in figure
Really, due in the cell sample Bcl-12 indexs expression it is relatively low, do not shown in figure, 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 inventive method is little with WesternBlot testing results difference, can accurately react stress reaction
The change of albumen.
The detection of autophagy marker protein under the conditions of embodiment 6, Nature enemy
Using the detection method and WesternBlot detection methods of embodiment 3, by the liver cancer Hep after Nature enemy 12h
G2 cell samples are detected, using the liver cancer Hep G2 cell samples of non-Nature enemy as control.Stress protein chip results
Data carry out fluorescence signal reading with the software of analysis software Gene Pro 6.0, read the before processing for obtaining each index
Fluorescence signal value afterwards, is normalized using internal reference GAPDH as standard, and interpretation of result is carried out with Grapa6.0 softwares, and (t is examined
Test).
As a result signal is as shown in Fig. 2 A is the inventive method testing result in figure, and B is that WesternBlot detects knot in figure
Really, due in the cell sample remaining 4 indexs expression it is relatively low, do not shown in figure, 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 less, can accurately react the change of stress response protein with WesternBlot testing results difference.
Claims (10)
1. a kind of micro-array chip for detecting protein expression in cellular stress, it includes solid carrier and array type is fixed on
The independently specific antibody of stress reaction mark on solid carrier；Described stress reaction mark is following each categories
A class or multiclass for will albumen：Cell propagation marker protein, Apoptosis marker protein, cell autophagy marker protein, cell are moved
Move marker protein, protein translation marker protein, cell dryness marker protein, cellular stress marker protein.
2. micro-array chip according to claim 1, it is characterised in that：Described stress reaction mark is following all kinds of
A class or multiclass for marker protein, all kinds of marker proteins contain multiple independently marker proteins, wherein：
Cell propagation 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 dryness 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.
3. micro-array chip according to claim 1, it is characterised in that：Solid carrier is the slide that aldehyde radicalization is modified.
4. micro-array chip according to claim 1, it is characterised in that：Array type is gone back 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.
5. the microarray core of protein expression in a kind of detection cellular stress prepared as described in any one of Claims 1 to 4
The method of piece, comprises the following steps：
It it is 300~400 μm by distance between point, point sample amount is 350~450pL, and concentration is resisted for 300~500 μ g/mL specificity
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.
6. method according to claim 5, it is characterised in that：Aldehyde radicalization modification method be：By the slide cleaned up
With containing 4.0~6.0% (V/V) aminomethoxy silane ethanol solution handle 20~40min, dry, then with contain 1.5~
The PBS solution of 3.5% (V/V) glutaraldehyde handles 50~70min, dries, and obtains the slide of aldehyde radicalization modification.
7. a kind of kit for detecting the micro-array chip of protein expression in cellular stress, it is characterised in that：Containing having the right
It is required that the micro-array chip described in 1~4 any one.
8. kit according to claim 7, it is characterised in that：Also contain cell pyrolysis liquid, Tissue lysates, dilution
One or more in liquid, washing lotion, ELIAS secondary antibody, luminous substrate.
9. kit according to claim 8, it is characterised in that：The dilution is protein stabiliser；
And/or washing lotion is neutral buffered liquid；
And/or the secondary antibody that ELIAS secondary antibody is horseradish peroxidase-labeled；
And/or luminous substrate is fluorescein 555.
10. the detection method of protein expression, comprises the following steps in cellular stress：
Cell sample to be checked is cracked, protein lysate is obtained, point sample is in micro- battle array described in any one of Claims 1 to 4
On row chip, it is incubated, cleaning；ELIAS secondary antibody is added, is incubated, cleaning；Luminous substrate is added, is incubated, cleaning；Read fluorescence
Signal, using positive control albumen as internal reference, is normalized by standard of internal reference, obtains half-quantitative detection result.
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Denomination of invention: A microarray chip, preparation method, kit and detection method for detecting protein expression in cell stress response Effective date of registration: 20210126 Granted publication date: 20190611 Pledgee: Bank of China Limited by Share Ltd. Guangzhou Tianhe branch Pledgor: GUANGDONG LONGSEE MEDICAL TECHNOLOGY Co.,Ltd. Registration number: Y2021440000021 |