CN107024588B - Detect the protein chip and kit of protein Acetylation Level - Google Patents
Detect the protein chip and kit of protein Acetylation Level Download PDFInfo
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- CN107024588B CN107024588B CN201610070351.9A CN201610070351A CN107024588B CN 107024588 B CN107024588 B CN 107024588B CN 201610070351 A CN201610070351 A CN 201610070351A CN 107024588 B CN107024588 B CN 107024588B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N2440/10—Post-translational modifications [PTMs] in chemical analysis of biological material acylation, e.g. acetylation, formylation, lipoylation, myristoylation, palmitoylation
Abstract
The present invention discloses a kind of protein chip and kit; it can detecte Acetylation Level after protein translation; the protein chip includes 68 strain specific antibodies; the kit includes the protein chip that the point is formed with 68 strain specific antibodies, further includes detection secondary antibody, the tyrasamine of biotin labeling, amplification of signal buffer, 30%H of anti-acetylated lysine antibody, HRP label2O2, fluorescence coupling Streptavidin, protein lysate, NHS- biotin, protein labeling buffer, label stop buffer, super filter tube.Detection method are as follows: take a part to carry out biotin labeling after collecting albumen sample; a part does not mark biotin; then hybridize respectively in two reaction chambers of protein chip; biotin labeling pattern detection albumen background level; the pattern detection protein acetylation level not marked; this sample acetylation signal/background signal ratio is obtained after acquiring the fluorescence signal of the two, analyzes Acetylation Level difference by comparing ratio difference between different samples.
Description
Technical field
The present invention relates to protein chip and kits, and acetylation is repaired after being able to detect protein translation more particularly to one kind
Adorn the protein chip of level and kit and detection method comprising the protein chip.
Background technique
With in for the first time discovery of the scientists to acetylation of histone such as Phillips, Allfrey sixties in last century,
In past 50 years, the mankind have significant progress to the understanding of protein acetylation.Protein acetylation modification is divided into two kinds,
It is a kind of for protein translation when N-terminal acetylation modification, all have an impact to the synthesis, positioning and stability of albumen.Another kind is
Acetylation modification after protein translation on lysine residue epsilon-amino, under acetyltransferase (HAT) catalysis, acetylcoenzyme
The acetyl group of A is transferred to the lysine of destination protein, this process is simultaneously also by histon deacetylase (HDAC) (HDAC)
Reversible adjusting.Acetylation is the important component of protein post-translational modification, and acetylation of histone can promote chromosome
Superhelix is opened, genetic transcription is promoted.By proteomics the study found that other than histone, in cytoplasm and
There is a large amount of protein acetylation in mitochondria, has participated in RNA shearing, cell cycle, DNA replication dna, transcription, nucleus
In a variety of bioprocess such as transhipment.In addition a large amount of intermediate supersession enzymes can be acetylation modification, and Dynamic Regulating Process is to control
Cell metabolism processed plays a significant role.The adjusting of Acetylation Level is out of control to can result in a variety of diseases, the second of a variety of transcription factors
Acylated level all affects the occurrence and development of cancer (including P53, STAT3, HIF-1 α etc.), and there are many be with HDAC at present
The clinical medicine of target spot such as SAHA (Vorinostat) is used for the treatment of cancer-related diseases.Therefore, how efficiently to detect
The Acetylation Level of protein is all significant in scientific research, medical diagnosis and pharmaceutical developments field in sample.
Although lysine acetylation plays a significant role as a kind of common posttranslational modification, due to relatively low
Abundance level, Acetylation Level detection needs acetylated protein is enriched in total protein.However acetylation antibody
It is lower to the compatibility of acetylated protein, it is not able to satisfy and directly acetylated protein is enriched with, but need proteopepsis
After peptide fragment, reuse antibody enrichment acetylation peptide fragment, means by mass spectral analysis detect.It is well known that mass spectrum
Analysis needs to carry out fragmentation processing to sample, this preprocessing process is extremely complex, needs to take a substantial amount of time and manpower,
It is with high costs, while mass spectral analysis is also higher to the demand of sample, and a small amount of sample clinically also limits mass-spectrometric technique
Utilization.
Protein chip is a kind of protein-function assays technology of high throughput, is the biological detection skill that developed recently gets up
Art.Using this technology, by lot of antibodies or the intensive place system of antigen on solid phase carrier, are had can detect simultaneously it is a variety of
Protein and the small feature of sample requirement amount, have very important significance for the research of high throughput gene expression.But mesh
It is preceding still not about for detecting the relevant technologies such as protein chip and kit of acetylation modification level after protein translation
It is open.
Inventor has developed a kind of protein chip according to many years research experience in protein chip field, can
It is horizontal for acetylation modification after detecting protein translation, while also disclosing the preparation method of the protein chip, application, reagent
The relevant technologies such as box and detection method, the above-mentioned technology of inventor's exploitation has been filled up repairs for acetylation after detecting protein translation
Adorn the technological gap in horizontal protein chip field.
Summary of the invention
The first technical problem to be solved by the present invention is, provides a kind of protein chip, can be simultaneously in test sample
Acetylation modification is horizontal after multiple protein translations.
The second technical problem to be solved by the present invention is, provides the application of the protein chip.
The third technical problem to be solved by the present invention is, provides a kind of kit, includes the protein chip, can
Acetylation modification is horizontal after detecting protein translation.
The fourth technical problem to be solved by the present invention is, provides the kit for detecting second after protein translation
The horizontal detection method of acylated modification.
One of to solve above-mentioned technical problem, protein chip provided by the invention is caught including what substrate and array were distributed
Antibody is obtained, the capture antibody includes but is not limited to following 68 strain specific antibodies: AML1, AMPK, APE, AR, α-Tubulin,
β-Catenin,Bcl-6,Beclin,BRAF,MYB,c-Myc,CREB,CTBP2,DEK,E2F1,E2F2,E2F3,EGFR,
Her2,Her4,ER,EKLF,FEN1,FOXO1,FOXO3,FOXO4,GATA1,GATA2,GATA3,GATA4,GR,HIF-1α,
HMGA1,HMGB1,HSP90,IRF2,IRS1,KRAS,Ku70,MDM2,MEF2A,NEIL2,NFκB,Notch1,P21,P53,
P65,PGC1,PTEN,RB1,KPNA2,RUNX2,SMAD2,SMAD4,SMAD7,SOX4,SOX9,SREBP1,SRY,STAT1,
STAT2,STAT3,TDG,VEGF,WRN,β-Actin,GAPDH,BSA。
The capture antibody is following 68 strain specific antibodies: AML1, AMPK, APE, AR, α-Tubulin, β-
Catenin,Bcl-6,Beclin,BRAF,MYB,c-Myc,CREB,CTBP2,DEK,E2F1,E2F2,E2F3,EGFR,Her2,
Her4,ER,EKLF,FEN1,FOXO1,FOXO3,FOXO4,GATA1,GATA2,GATA3,GATA4,GR,HIF-1α,HMGA1,
HMGB1,HSP90,IRF2,IRS1,KRAS,Ku70,MDM2,MEF2A,NEIL2,NFκB,Notch1,P21,P53,P65,
PGC1,PTEN,RB1,KPNA2,RUNX2,SMAD2,SMAD4,SMAD7,SOX4,SOX9,SREBP1,SRY,STAT1,STAT2,
STAT3,TDG,VEGF,WRN,β-Actin,GAPDH,BSA。
The substrate includes being modified or modified glass slide, plastic slide, diaphragm etc. can be used for protein site
The material of system.
The protein chip, further includes positive control and negative control, and the positive control is the BSA of biotin labeling
(BSA- biotin) and acetylation BSA (Ac-BSA), the negative control be BSA (bovine serum albumin(BSA)), mouse IgG and
rabbit normal IgG。
The protein chip is that the capture antibody, positive control and negative control are selected and be formed on the substrate respectively
It is manufactured.
Point makes 3 repetition points on chip for the positive control, negative control and each capture antibody.
Above-mentioned 68 strain specific antibodies is by well-chosen.To realize efficient while detecting multiple proteins
Acetylation Level, inventor have made intensive studies a large amount of possible antibody that protein acetylation occurs, have filtered out
Stating 68 kinds can occur that obvious acetylation modification, atopic are good, detection signal is obvious and signal and antigen concentration are in preferably
The specific antibody of linear relationship, 68 strain specific antibodies disclosed by the invention may be advantageously employed in protein acetylation modification
Horizontal detection, testing result can reaction detection sample on the whole protein acetylation modification it is horizontal.It relies on simultaneously
Above-mentioned 68 strain specific antibodies can be directed to the needs of different experiments, and protein acetyl can potentially be occurred by increasing and putting system
Change or need to examine other whether changed different antibody of protein acetylation modification level, it is personalized fixed to can satisfy
The requirement of system.
To solve above-mentioned technical problem two, invention additionally discloses the protein chips to be used to prepare detection protein translation
The application of the device of acetylation modification level afterwards.Protein chip of the invention can be used for preparing acetylation after detection protein translation
Horizontal device is modified, such as kit etc. meets the use demand in fields such as scientific research, medical diagnosis and pharmaceutical developments.
To solve above-mentioned technical problem three, kit provided by the invention, including the protein chip.
The kit further includes the detection two of anti-acetylated lysine antibody, HRP (horseradish peroxidase) label
Anti-, the tyrasamine of biotin labeling, amplification of signal buffer, 30%H2O2, fluorescence be coupled Streptavidin, protein lysate, NHS-
Biotin, protein labeling buffer, label stop buffer and super filter tube.
To solve above-mentioned technical problem four, the kit provided by the invention is for detecting acetyl after protein translation
It is following (its testing process is as shown in Figure 1) to change the horizontal detection method of modification:
1, the preparation of sample
1. test sample: taking cell or tissue sample, protein lysate is added, is sufficiently centrifuged after reaction, collects supernatant
And it is quantified to get to test sample (for detecting acetylation signal)；
2. background sample: taking part test sample, protein labeling buffer is added and mixes, NHS- biotin is added and is marked
Note, add label stop buffer end mark, then be added 1xPBS (phosphate buffered saline solution), be put into super filter tube, through from
Take ultrafiltrate to save after the heart to get to background sample (for detecting albumen background signal).
2, protein chip hybridizes
1. first closing protein chip with BSA, the test sample and background sample of equivalent are then taken, in protein chip
It is separately added into taken test sample and background sample in two reaction chambers, it is subjected to hybridization incubation with protein chip, clearly
It washes；
2. detection group: continuously adding anti-acetylated lysine antibody in the reaction chamber, be incubated for, HRP label is added in cleaning
Secondary antibody is detected, is incubated for, cleaning is added the tyrasamine reaction of amplification of signal buffer and biotin labeling, finally adds fluorescence idol
Join Streptavidin, place, cleans, drying；
3. background group: fluorescence is added in the reaction chamber and is coupled Streptavidin, places, cleans, drying.
3, data collection and analysis
The fluorescence signal that detection group and background group are read using chip scanner, with 6.0 software of Genepix Pro to obtaining
The fluorescence signal obtained is analyzed, and is acquired the fluorescence signal median in each capture antibody dots and is calculated repetition point with this
Average value and CV value.Unlabelled protein sample and anti-acetylated lysine antibody are added in reaction chamber, what scanning obtained
Signal is acetylation signal, and the protein sample of biotin labeling is added in reaction chamber, scanning obtained signal is
Albumen background signal.The Acetylation Level of sample protein acetylation signal/The ratio of background signal indicates that the ratio is bigger
Then illustrate that the Acetylation Level of sample protein is higher.
The difference of Acetylation Level is analyzed according to acetylation signal/background signal ratio between sample.
The present invention provide for the first time it is a kind of can simultaneously in test sample multiple protein acetylation modification levels albumen
Chip and kit, compared to the prior art in using mass spectral analysis detection protein Acetylation Level method, the present invention without
The preprocessing process such as complicated digestion purifying need to be carried out to protein, and it is time-consuming to can solve detection protein acetylation modification at present
Laborious problem, and multiple proteins that can simultaneously in test sample, detect more comprehensive, and detection efficiency is high, and sample needs
The amount of asking is small, has very important significance for the research of high throughput gene expression, while relying on this platform, can be for difference
The needs point of experiment makes different antibody, can satisfy the requirement of personalized customization.
Detailed description of the invention
Fig. 1 is acetylated protein chip operation flow chart.
Fig. 2 protein chip detects the acetylation variation of TSA inducing cell.
Fig. 3 protein chip detects the acetylation variation of P300 overexpressing cell.
Specific embodiment
Clear, complete description is carried out to technical solution of the present invention below in conjunction with experimental data and attached drawing, it is clear that institute
The embodiment of description is a part of embodiment of the invention, rather than whole embodiments.Based on the implementation in the present invention
Example, those of ordinary skill in the art's every other embodiment obtained without making creative work, all
Belong to the scope of protection of the invention.
Embodiment one
Protein chip the preparation method is as follows:
68 strain specific antibodies are diluted to 0.5 μ g/ μ l with PBS-15% glycerol, using chip point platform by antibody by
A little in system to chip.500 μm of spacing of 200 μm of each spot diameter, point and point；Use BSA (the BSA- biology of biotin labeling
Element), acetylation BSA (Ac-BSA) be used as positive control, use BSA, mouse IgG, rabbit normal IgG as negative
Control；It is put into 384 orifice plates according to certain sequence, carries out chip point system, sun ginseng, yin ginseng and capture antibody using chip point sample instrument
3 repetition points of point system, are prepared into protein-chip.68 strain specific antibodies are as follows: AML1, AMPK, APE, AR, α-
Tubulin,β-Catenin,Bcl-6,Beclin,BRAF,MYB,c-Myc,CREB,CTBP2,DEK,E2F1,E2F2,E2F3,
EGFR,Her2,Her4,ER,EKLF,FEN1,FOXO1,FOXO3,FOXO4,GATA1,GATA2,GATA3,GATA4,GR,HIF-
1α,HMGA1,HMGB1,HSP90,IRF2,IRS1,KRAS,Ku70,MDM2,MEF2A,NEIL2,NFκB,Notch1,P21,
P53,P65,PGC1,PTEN,RB1,KPNA2,RUNX2,SMAD2,SMAD4,SMAD7,SOX4,SOX9,SREBP1,SRY,
STAT1,STAT2,STAT3,TDG,VEGF,WRN,β-Actin,GAPDH,BSA。
Specific antibody is can be directed to the needs of different experiments by well-chosen, rely on embodiment in above-mentioned 68
One protein chip, the targeted different antibody of point fixture, and it is used for the protein Acetylation Level of test sample, it can
Meet the requirement of personalized customization.
Embodiment two:
The detection method of the kit of protein chip based on embodiment one:
1, the preparation of sample
1. test sample: taking 106–5x 106100-200 μ l protein lysates are added in cell or 10-40mg tissue samples,
It places 30 minutes at 4 DEG C and vibrates frequently, then 10000g is centrifuged 15 minutes at 4 DEG C, and it collects supernatant and is quantified,
Obtain test sample (for detecting acetylation signal)；
2. background sample: taking 100 μ g test samples, protein labeling buffer is added and mixes to 50 μ l, 2 μ l NHS- are added
Biotin reacts 2 hours at room temperature to be marked, and is added 1.6 μ l label stop buffer and was terminated in room temperature reaction 30 minutes
Label, is then added 450 μ l 1xPBS, is put into super filter tube, takes ultrafiltrate to protect in -20 DEG C after 4 DEG C of 10000g are centrifuged 30 minutes
It deposits to get to blank sample (for detecting albumen background signal).
2, protein chip hybridizes
1. protein chip 1%BSA room temperature is first closed 1h, the test sample and blank sample of equivalent are then taken,
It is separately added into taken test sample and blank sample in two reaction chambers of protein chip, by itself and protein chip at 37 DEG C
It is cleaned after carrying out hybridization incubation 1.5 hours；
2. detection group: anti-acetylated lysine antibody is continuously added in the reaction chamber, 37 DEG C are incubated for 1 hour, PBST cleaning,
The detection secondary antibody of HRP label is added, is incubated at room temperature 1 hour, after PBST cleaning, addition contains 0.0015%H2O2Amplification of signal is slow
The tyrasamine of fliud flushing and biotin labeling reacts at room temperature 10 minutes, finally adds 0.1 μ g/ml fluorescence coupling Streptavidin, room
Temperature places 30min, cleans, drying；
3. background group: 0.1 μ g/ml fluorescence coupling Streptavidin is added in the reaction chamber, is placed at room temperature for 30min, cleans,
Drying；
All cleaning processes all use PBST to clean 3 times, every time 5 minutes.
3, data collection and analysis
The fluorescence signal that detection group and blank group are read using chip scanner, with 6.0 software of Genepix Pro to obtaining
Fluorescence signal analyzed, acquire the median in each capture antibody dots and with this calculate repetition point average value and
CV value.Unlabelled protein sample and anti-acetylated lysine antibody are added in reaction chamber, scanning obtained signal is
Acetylation signal, and the protein sample of biotin labeling is added in reaction chamber, scanning obtained signal is albumen background
Signal.The Acetylation Level of test sample albumen indicates that the ratio the big with acetylation signal/background signal ratio, says
The Acetylation Level of bright sample protein is higher, and the difference of Acetylation Level can be by comparing different samples between different samples
Acetylation signal/background signal ratio is analyzed.
Embodiment three
TSA (Trichostatin A) handles cell model
1, experimental principle
TSA (Trichostatin A) is a kind of reversible HDAC (histone deacetylase, histone deacetylase
Change enzyme) inhibitor, the Acetylation Level by inhibiting the process of albumen deacetylate group, in raising cell.With DMSO and TSA points
Sample is managed in other places, and DMSO processing is check sample, is compared and is passed through after TSA is handled with protein chip of the invention and kit detection
The variation of Acetylation Level in DMSO treated cell, to illustrate detection effect of the invention.
2, sample preparation
Gastric cancer cell MKN45 is in 1640 culture medium of RAPI for containing 10%FBS (fetal calf serum, fetal calf serum)
Middle culture collects cell protein after being handled 18 hours with DMSO and 3 μM of TSA respectively to 70% density.
3, test method
A, 10 are taken6100 μ l protein lysate, 4 DEG C of cracking 30min are added in the MKN45 cell handled through DMSO and TSA,
Supernatant is collected after 10000g centrifugation 15min, measures concentration, wherein 100 μ g albumen progress biotin labeling is taken respectively, for detecting
Albumen background signal, remaining is for detecting acetylation signal；
B, it takes out protein chip and closes 1h with 1%BSA room temperature, take the DMSO processing group and TSA processing group of equivalent respectively
Test sample and background sample are added in two reaction chambers of difference of chip, 37 DEG C after progress hybridization incubation 1.5 hours it is clear
It washes；
C, in detection group reaction chamber, anti-acetylated lysine antibody is continuously added, 37 DEG C are incubated for 1 hour, PBST cleaning,
The detection secondary antibody of HRP label is added, is incubated at room temperature 1 hour, after PBST cleaning, addition contains 0.0015%H2O2Amplification of signal is slow
The tyrasamine of fliud flushing and biotin labeling reacts at room temperature 10 minutes, finally adds 0.1 μ g/ml fluorescence coupling Streptavidin, room
Temperature places 30min, cleans, drying；0.1 μ g/ml fluorescence coupling Streptavidin is added in background group reaction chamber, is placed at room temperature for
30min is cleaned, drying；
D, DMSO processing group and TSA processing group sample fluorescence signal are read using chip scanner, with Genepix Pro
6.0 softwares analyze it, and compare acetylation signal/background signal ratio between DMSO processing and two groups of samples of TSA processing group
The difference of value.
4, experimental result
MKN45 cell is handled by TSA, by WB it can be found that Acetylation Level significantly improves (Fig. 2 a).We are with this
The cell of TSA induction is model, and the acetylation between check sample (Control group) and TSA processing sample is detected with the present invention
Difference, the Acetylation Level by comparing discovery GATA1 and Histone (histone) albumen have relatively apparent rising, respectively
1.6 times and 3.1 times (Fig. 2 b, 2c) are raised.Experimental result is consistent with expection, illustrates that protein chip and kit of the invention can
With horizontal for detecting protein acetylation modification well.
Embodiment 4
P300 overexpressing cell model inspection
1, experimental principle
P300 is a kind of acetyltransferase (HAT), and substrate includes nearly all histone and a large amount of non-group of egg
It is white, the variation of cellular acetylation level can be detected from other side by being overexpressed P300, to be subject to protein chip
Verifying.
2, sample preparation
Stomach cancer cell AGS is cultivated in 1640 culture medium of RAPI containing 10%FBS to 70% density, is used
Lipofectamine2000 distinguishes Transfection of GFP (Green Fluorescent Protein, green fluorescent protein) and P300 matter
Grain, transfection collected cell RNA and protein after 48 hours respectively.RNA is overexpressed P300 water to detect cell after reverse transcription
It is flat, albumen to hybridization check Acetylation Level of the present invention.
3, test method
A, RNA extract, reverse transcription and quantitative:
10 are taken respectively51ml Trizol lysate, RNA extraction process is added in blank and the MKN45 cell for being overexpressed P300
It is operated according to Trizol specification, reverse transcription and fluorescent quantitation are according to Takara reverse transcription reagent box and SYBR fluorescent quantitation
Kit specification is operated.
B, protein is collected, chip hybridization detects:
B1. 10 are taken54 DEG C of cracking 30min, 10000g centrifugations of lysate are added in blank and the MKN45 cell for being overexpressed P300
Supernatant is collected after 15min, carries out biotin labeling, and for detecting albumen background signal, remaining is for detecting acetylation signal；
B2. protein chip is taken out, takes equivalent detection sample and background sample to be added in the reaction chamber of chip respectively, 37
DEG C carry out hybridization incubation 1.5 hours after clean；
B3. in detection group reaction chamber, anti-acetylated lysine antibody is continuously added, 37 DEG C are incubated for 1 hour, and PBST is clear
It washes, the detection secondary antibody of HRP label is added, be incubated at room temperature 1 hour, after PBST cleaning, addition contains 0.0015%H2O2Amplification of signal
The tyrasamine of buffer and biotin labeling reacts at room temperature 10 minutes, finally adds 0.1 μ g/ml fluorescence coupling Streptavidin,
It is placed at room temperature for 30min, is cleaned, drying；0.1 μ g/ml fluorescence coupling Streptavidin is added in background group reaction chamber, room temperature is put
30min is set, is cleaned, drying；
B4. blanc cell is read using chip scanner and be overexpressed the fluorescence signal of P300 cell sample, use Genepix
Pro6.0 software analyzes it, and compares blanc cell and is overexpressed acetylation signal/background signal ratio between P300 cell
The difference of value.
4, experimental result
P300mRNA significantly rises (Fig. 3 a) after cell transfecting P300 plasmid 48 hours, after being detected with protein chip, discovery
Compared with check sample (Control group), P300 overexpressing cell model includes Histone, GATA1, EKLF, Tubulin
Acetylation Level up-regulation (Fig. 3 b, 3c) all has occurred in multiple albumen such as (tubulin), and wherein GATA1 and Tubulin are raised
1.6 times, Histone and EKLF raised 1.3 times.Experimental result is consistent with expection, illustrates protein chip and examination of the invention
It is horizontal that agent box may be advantageously employed in detection protein acetylation modification.
In conclusion the various embodiments described above and attached drawing are only part preferred embodiment of the invention, not to limit
Determine protection scope of the present invention, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done,
It should all be included within the scope of the present invention.
Claims (10)
1. a kind of protein chip, which is characterized in that be distributed in the capture antibody on substrate, the capture including substrate and array
Antibody includes following 68 strain specific antibodies: AML1, AMPK, APE, AR, α-Tubulin, β-Catenin, Bcl-6, Beclin,
BRAF,MYB,c-Myc,CREB,CTBP2,DEK,E2F1,E2F2,E2F3,EGFR,Her2,Her4,ER,EKLF,FEN1,
FOXO1,FOXO3,FOXO4,GATA1,GATA2,GATA3,GATA4,GR,HIF-1α,HMGA1,HMGB1,HSP90,IRF2,
IRS1,KRAS,Ku70,MDM2,MEF2A,NEIL2,NFκB,Notch1,P21,P53,P65,PGC1,PTEN,RB1,KPNA2,
RUNX2,SMAD2,SMAD4,SMAD7,SOX4,SOX9,SREBP1,SRY,STAT1,STAT2,STAT3,TDG,VEGF,WRN,
β-Actin,GAPDH,BSA；The protein chip further includes positive control and negative control, and the positive control is biotin mark
The BSA and acetylation BSA of note, the negative control are BSA, mouse IgG and rabbit normal IgG.
2. protein chip according to claim 1, which is characterized in that the capture antibody is anti-for following 68 species specificity
Body: AML1, AMPK, APE, AR, α-Tubulin, β-Catenin, Bcl-6, Beclin, BRAF, MYB, c-Myc, CREB,
CTBP2,DEK,E2F1,E2F2,E2F3,EGFR,Her2,Her4,ER,EKLF,FEN1,FOXO1,FOXO3,FOXO4,GATA1,
GATA2,GATA3,GATA4,GR,HIF-1α,HMGA1,HMGB1,HSP90,IRF2,IRS1,KRAS,Ku70,MDM2,MEF2A,
NEIL2,NFκB,Notch1,P21,P53,P65,PGC1,PTEN,RB1,KPNA2,RUNX2,SMAD2,SMAD4,SMAD7,
SOX4,SOX9,SREBP1,SRY,STAT1,STAT2,STAT3,TDG,VEGF,WRN,β-Actin,GAPDH,BSA。
3. protein chip according to claim 1 or 2, which is characterized in that the substrate be glass slide, plastic slide or
Diaphragm.
4. protein chip according to claim 1, which is characterized in that the protein chip is respectively that the capture is anti-
Made of body, positive control and negative control are selected and are formed on the substrate.
5. protein chip according to claim 4, which is characterized in that the positive control, negative control and each catch
It obtains antibody and puts at least three repetition point processed on chip.
6. a kind of protein chip is used to prepare the application of the device of acetylation modification level after detection protein translation, feature exists
Include substrate in, the protein chip and array is distributed in capture antibody on substrate, the capture antibody includes following 68
Strain specific antibodies: AML1, AMPK, APE, AR, α-Tubulin, β-Catenin, Bcl-6, Beclin, BRAF, MYB, c-
Myc,CREB,CTBP2,DEK,E2F1,E2F2,E2F3,EGFR,Her2,Her4,ER,EKLF,FEN1,FOXO1,FOXO3,
FOXO4,GATA1,GATA2,GATA3,GATA4,GR,HIF-1α,HMGA1,HMGB1,HSP90,IRF2,IRS1,KRAS,
Ku70,MDM2,MEF2A,NEIL2,NFκB,Notch1,P21,P53,P65,PGC1,PTEN,RB1,KPNA2,RUNX2,
SMAD2,SMAD4,SMAD7,SOX4,SOX9,SREBP1,SRY,STAT1,STAT2,STAT3,TDG,VEGF,WRN,β-
Actin,GAPDH,BSA。
7. application as claimed in claim 6, which is characterized in that the capture antibody is following 68 strain specific antibodies: AML1,
AMPK,APE,AR,α-Tubulin,β-Catenin,Bcl-6,Beclin,BRAF,MYB,c-Myc,CREB,CTBP2,DEK,
E2F1,E2F2,E2F3,EGFR,Her2,Her4,ER,EKLF,FEN1,FOXO1,FOXO3,FOXO4,GATA1,GATA2,
GATA3,GATA4,GR,HIF-1α,HMGA1,HMGB1,HSP90,IRF2,IRS1,KRAS,Ku70,MDM2,MEF2A,NEIL2,
NFκB,Notch1,P21,P53,P65,PGC1,PTEN,RB1,KPNA2,RUNX2,SMAD2,SMAD4,SMAD7,SOX4,
SOX9,SREBP1,SRY,STAT1,STAT2,STAT3,TDG,VEGF,WRN,β-Actin,GAPDH,BSA。
8. a kind of kit, which is characterized in that the kit includes protein chip of any of claims 1 or 2.
9. kit according to claim 8, which is characterized in that the kit further includes that anti-acetylated lysine is anti-
Detection secondary antibody, the tyrasamine of biotin labeling, amplification of signal buffer, 30%H of body, HRP label2O2, fluorescence coupling strepto- it is affine
Element, protein lysate, NHS- biotin, protein labeling buffer, label stop buffer and super filter tube.
10. the detection method of Acetylation Level after a kind of detection protein translation, which is characterized in that the detection method right to use
Benefit require 9 described in kit, and the following steps are included:
The preparation of step 1, sample:
1. test sample: taking cell or tissue sample, protein lysate is added, is sufficiently centrifuged after reaction, collects supernatant and goes forward side by side
It is capable to quantify to get test sample is arrived；
2. background sample: part test sample is taken, protein labeling buffer is added and mixes, NHS- biotin is added and is marked,
Label stop buffer end mark is added, 1xPBS is then added, is put into super filter tube, takes ultrafiltrate to save after being centrifuged, i.e.,
Obtain background sample；
Step 2, protein chip hybridization:
1. first closing protein chip with BSA, the test sample and background sample of equivalent are then taken, at two of protein chip
It is separately added into taken test sample and background sample in reaction chamber, it is subjected to hybridization incubation, cleaning with protein chip；
2. detection group: continuously adding anti-acetylated lysine antibody in the reaction chamber, be incubated for, the detection of HRP label is added in cleaning
Secondary antibody is incubated for, cleaning, and the tyrasamine reaction of amplification of signal buffer and biotin labeling is added, and finally adds fluorescence coupling chain
Mould Avidin is placed, and is cleaned, drying；
3. background group: fluorescence is added in the reaction chamber and is coupled Streptavidin, places, cleans, drying；
Step 3, data collection and analysis:
The fluorescence signal that detection group and background group are read using chip scanner, is divided with fluorescence signal of the software to acquisition
Analysis, is acquired the median in each capture antibody dots and is calculated the average value and CV value of repetition point with this；It is added in reaction chamber
Unlabelled protein sample and anti-acetylated lysine antibody, scanning obtained signal is acetylation signal, and is reacted
The protein sample of biotin labeling is added in room, scanning obtained signal is albumen background signal；Test sample albumen
Acetylation Level indicated with acetylation signal/background signal ratio.
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| CN108690137A (en) * | 2018-05-31 | 2018-10-23 | 山东省医学科学院基础医学研究所 | M8Sac71 antibody and the preparation method and application thereof |
| CN110082327A (en) * | 2019-04-30 | 2019-08-02 | 阜阳师范学院 | A kind of egg mother cell H3K27 acetylation index analysis method |
| CN113155577B (en) * | 2021-05-08 | 2023-07-25 | 右江民族医学院 | Preparation method for identifying specific protein acetylation modified sample |
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