CN107024588A - 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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- CN107024588A CN107024588A CN201610070351.9A CN201610070351A CN107024588A CN 107024588 A CN107024588 A CN 107024588A CN 201610070351 A CN201610070351 A CN 201610070351A CN 107024588 A CN107024588 A CN 107024588A
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Abstract
The present invention discloses a kind of protein chip and kit; Acetylation Level after protein translation can be detected; the protein chip includes 68 strain specific antibodies; the kit is formed with the protein chip of 68 strain specific antibodies comprising the point, includes detection secondary antibody, the tyrasamine of biotin labeling, amplification of signal buffer solution, the 30%H of anti-acetylated lysine antibody, HRP mark2O2, fluorescence coupling Streptavidin, protein lysate, NHS- biotins, protein labeling buffer solution, mark stop buffer, super filter tube.Detection method is：Collect and take a part to carry out biotin labeling after albumen sample; a part does not mark biotin; then hybridize respectively in two reative cells 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 both fluorescence signals of collection, Acetylation Level difference is analyzed by ratio difference between relatively more different samples.
Description
Technical field
The present invention relates to protein chip and kit, more particularly to one kind can detect acetylation modification level after protein translation
Protein chip and kit and detection method comprising the protein chip.
Background technology
With in the first discovery of the scientist to acetylation of histone such as Phillips, Allfrey sixties in last century, past 50
Nian Zhong, understanding of the mankind to protein acetylation has significant progress.Protein acetylation modification is divided into two kinds, and one kind is egg
The acetylation modification of N-terminal during white translation, synthesis, positioning and stability to albumen all have an impact.Another is protein translation
Acetylation modification on lysine residue epsilon-amino afterwards, under acetyltransferase (HAT) catalysis, the second of acetyl coenzyme A
Acyl group group is transferred to the lysine of destination protein, and this process simultaneously also can by histon deacetylase (HDAC) (HDAC)
Inverse regulation.Acetylation is the important component of protein post-translational modification, and it is super that acetylation of histone can promote chromosome to open
Helical structure, promotes genetic transcription.Found by the research of proteomics, in addition to histone, in cytoplasm and mitochondria
In there is substantial amounts of protein acetylation, participated in RNA shearings, cell cycle, DNA replication dna, transcription, cell consideration convey
In a variety of bioprocess such as fortune.A large amount of intermediate supersession enzymes can be acetylation modification in addition, and its Dynamic Regulating Process is thin to control
Born of the same parents' metabolism plays an important roll.The regulation of Acetylation Level is out of control to can result in a variety of diseases, the acetylation water of a variety of transcription factors
Flat (including P53, STAT3, HIF-1 α etc.) all affects the occurrence and development of cancer, existing at present a variety of using HDAC as target
The clinical medicine such as SAHA (Vorinostat) of point 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.
It is due to relatively low abundance although lysine acetylation plays an important roll as a kind of common posttranslational modification
Level, the detection of Acetylation Level needs to be enriched with acetylated protein in total protein.But acetylation antibody is to acetylation
The compatibility of albumen is relatively low, it is impossible to which satisfaction is directly enriched with to acetylated protein, but needs proteopepsis into after peptide fragment,
Reuse antibody enrichment acetylation peptide fragment, means by mass spectral analysis are detected.It is well known that mass spectral analysis needs pair
Sample carries out fragmentation processing, and this preprocessing process is extremely complex, it is necessary to take a substantial amount of time and manpower, with high costs,
Mass spectral analysis simultaneously is also higher to the demand of sample, and a small amount of sample clinically also limit the utilization of mass-spectrometric technique.
Protein chip is a kind of high-throughout protein-function assays technology, is the Measurement for Biotechnique that developed recently gets up.Profit
With this technology, by by lot of antibodies or the intensive place system of antigen on solid phase carrier, with multiple proteins can be detected simultaneously
And sample requirement amount it is small the characteristics of, the research for high flux gene expression has very important significance.But, do not have still at present
It is related to the disclosure for detecting the correlation technique such as protein chip and kit of acetylation modification level after protein translation.
Inventor have developed a kind of protein chip according to the research experience for many years in protein chip field, can be used in inspection
Acetylation modification level after protein translation is surveyed, while also disclosing the preparation method of the protein chip, using, kit and inspection
The correlation techniques such as survey method, the above-mentioned technology of inventor's exploitation has been filled up for detecting acetylation modification level after protein translation
The technological gap in protein chip field.
The content of the invention
One of technical problems to be solved by the invention are there is provided a kind of protein chip, and multiple eggs in sample can be detected simultaneously
Acetylation modification level after white matter translation.
The two of the technical problems to be solved by the invention are that there is provided the application of the protein chip.
The three of the technical problems to be solved by the invention are there is provided a kind of kit, comprising the protein chip, can detect
Acetylation modification level after protein translation.
The four of the technical problems to be solved by the invention are to be used to detect that acetylation is repaiied after protein translation there is provided the kit
The detection method of decorations level.
To solve the capture antibody of one of above-mentioned technical problem, the protein chip that the present invention is provided, including substrate and array distribution,
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 can be used for protein site through modification or modified glass slide, plastic slide, diaphragm etc.
Material.
The protein chip, in addition to positive control and negative control, the positive control are the BSA (BSA- of biotin labeling
Biotin) and acetylation BSA (Ac-BSA), the negative control is BSA (bovine serum albumin(BSA)), mouse IgG and rabbit
normal IgG。
The protein chip, the capture antibody, positive control and negative control respectively selected to be formed on the substrate and be made.
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 the acetylation of multiple proteins
Level, inventor has made intensive studies to the substantial amounts of antibody that may occur protein acetylation, and having filtered out above-mentioned 68 kinds can
Occur good obvious acetylation modification, atopic, obvious detection signal and signal and antigen concentration are in preferable linear relationship
Specific antibody, 68 strain specific antibodies disclosed by the invention may be advantageously employed in the detection of protein acetylation modification level,
Its testing result can reaction detection sample on the whole protein acetylation modification level.Rely on above-mentioned 68 species specificity simultaneously
Antibody, the need for being directed to different experiments, protein acetylation can potentially be occurred or need to examine albumen by increasing and putting system
Other the different antibody whether matter acetylation modification level changes, disclosure satisfy that the requirement of personalized customization.
To solve the two of above-mentioned technical problem, it is used to prepare acetyl after detection protein translation invention additionally discloses the protein chip
Change the application of the device of modification level.The protein chip of the present invention can be used for preparing acetylation modification level after detection protein translation
Device, such as kit, meet the fields such as scientific research, medical diagnosis and pharmaceutical developments use demand.
To solve the three of above-mentioned technical problem, the kit that provides of the present invention, including described protein chip.
The kit, in addition to anti-acetylated lysine antibody, HRP (horseradish peroxidase) mark detection secondary antibody,
Tyrasamine, amplification of signal buffer solution, the 30%H of biotin labeling2O2, fluorescence coupling Streptavidin, protein lysate, NHS-
Biotin, protein labeling buffer solution, mark stop buffer and super filter tube.
To solve the four of above-mentioned technical problem, the kit that the present invention is provided is used to detect acetylation modification after protein translation
The detection method of level is following (its testing process is as shown in Figure 1)：
1st, the preparation of sample
1. sample is detected：Cell or tissue sample is taken, protein lysate is added, is fully centrifuged after reaction, collect supernatant and go forward side by side
Row is quantitative, that is, obtains detection sample (being used to detect acetylation signal)；
2. background sample：Take part to detect sample, add protein labeling buffer solution and mix, add NHS- biotins and be marked,
Mark stop buffer end mark is added, 1xPBS (phosphate buffered saline solution) is then added, is put into super filter tube, through centrifugation
After take ultrafiltrate to preserve, that is, obtain background sample (be used for detect albumen background signal).
2nd, protein chip hybridizes
1. first protein chip is closed with BSA, the detection sample and background sample of equivalent is then taken, at two of protein chip
Taken detection sample and background sample is separately added into reative cell, itself and protein chip are subjected to hybridization incubation, cleaning；
2. detection group：Anti- acetylated lysine antibody is continuously added in the reaction chamber, is incubated, and cleaning adds the inspection of HRP marks
Secondary antibody is surveyed, is incubated, cleaning adds the tyrasamine reaction of amplification of signal buffer solution and biotin labeling, finally adds fluorescence coupling
Streptavidin, is placed, and is cleaned, and is dried；
3. background group：Fluorescence coupling Streptavidin is added in the reaction chamber, is placed, is cleaned, dries.
3rd, data collection and analysis
The fluorescence signal of detection group and background group is read using chip scanner, with fluorescence of the softwares of Genepix Pro 6.0 to acquisition
Signal is analyzed, and gathers the fluorescence signal median in each capture antibody dots and the average value and CV of repetition point are calculated with this
Value.Unlabelled protein sample and anti-acetylated lysine antibody are added in reative cell, it scans obtained signal as acetyl
Change signal, and the protein sample of biotin labeling is added in reative cell, it is albumen background signal that it, which scans obtained signal,.
The Acetylation Level of sample protein acetylation signal/The ratio of background signal represents that the ratio is more big, illustrate sample protein
Acetylation Level is higher.
The difference of Acetylation Level is analyzed according to the ratio of acetylation signal/background signal between sample.
The present invention can detect the protein chip of multiple protein acetylation modification levels and examination in sample simultaneously there is provided a kind of first
Agent box, compared to the method for detecting protein Acetylation Level using mass spectral analysis in the prior art, the present invention is without to protein
The preprocessing process such as complicated digestion purifying are carried out, can solve to detect the problem of protein acetylation modification is wasted time and energy at present,
And the multiple proteins in sample can be detected simultaneously, detect more comprehensive, detection efficiency is high, and sample requirements are small, for
The research of high flux gene expression has very important significance, while relying on this platform, can be directed to point the need for different experiments
The different antibody of system, disclosure satisfy that the requirement of personalized customization.
Brief description of the drawings
Fig. 1 is acetylated protein chip operation flow chart.
The acetylation change of Fig. 2 protein chips detection TSA inducing cells.
The acetylation change of Fig. 3 protein chips detection P300 overexpressing cells.
Embodiment
Clear, complete description is carried out to technical scheme below in conjunction with experimental data and accompanying drawing, it is clear that described
Embodiment be the present invention a part of embodiment, rather than whole embodiment.Based on the embodiment in the present invention, sheet
The every other embodiment that field those of ordinary skill is obtained on the premise of creative work is not made, belongs to this hair
The scope of bright protection.
Embodiment one
The preparation method of protein chip is as follows：
68 strain specific antibodies are diluted to 0.5 μ g/ μ l with PBS-15% glycerine, antibody is put into system one by one using chip point platform
Onto chip.Each 200 μm of spot diameter, point and 500 μm of spacing of point；Using the BSA (BSA- biotins) of biotin labeling,
Acetylation BSA (Ac-BSA) is used as feminine gender as positive control using BSA, mouse IgG, rabbit normal IgG
Control；It is put into according to certain order in 384 orifice plates, chip point system is carried out using chip point sample instrument, sun ginseng, cloudy ginseng and capture is anti-
Body puts 3 repetition points of system, is prepared into protein-chip.68 strain specific antibodies is：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, by well-chosen, the need for being directed to different experiments, to rely on the egg of embodiment one in above-mentioned 68
White chip, point fixture targetedly different antibody, and for detecting the protein Acetylation Level of sample disclosure satisfy that
The requirement of propertyization customization.
Embodiment two：
The detection method of the kit of protein chip based on embodiment one：
1st, the preparation of sample
1. sample is detected：Take 106–5x 106Cell or 10-40mg tissue samples, add 100-200 μ l protein lysates,
Place 30 minutes and vibrate frequently at 4 DEG C, then 10000g is centrifuged 15 minutes at 4 DEG C, collect supernatant and determined
Amount, that is, obtain detection sample (being used to detect acetylation signal)；
2. background sample：Take 100 μ g to detect sample, add protein labeling buffer solution and mix to 50 μ l, add 2 μ l NHS- biological
Reaction is marked element for 2 hours at room temperature, adds 1.6 μ l mark stop buffers in 30 minutes end marks of room temperature reaction,
Then 450 μ l 1xPBS are added, super filter tube is put into, take ultrafiltrate to be preserved in -20 DEG C after being centrifuged 30 minutes through 4 DEG C of 10000g,
Obtain blank sample (being used to detect albumen background signal).
2nd, protein chip hybridizes
1. protein chip is first closed into 1h with 1%BSA room temperatures, the detection sample and blank sample of equivalent is then taken, in albumen
Taken detection sample and blank sample is separately added into two reative cells of chip, it is miscellaneous in 37 DEG C of progress with protein chip
Hand over and cleaned after being incubated 1.5 hours；
2. detection group：Anti- acetylated lysine antibody is continuously added in the reaction chamber, and 37 DEG C are incubated 1 hour, PBST cleanings, plus
Enter the detection secondary antibody of HRP marks, be incubated at room temperature 1 hour, after PBST cleanings, addition contains 0.0015%H2O2Amplification of signal
The tyrasamine of buffer solution and biotin labeling is reacted at room temperature 10 minutes, finally adds 0.1 μ g/ml fluorescence coupling Streptavidin,
Room temperature places 30min, cleans, and dries；
3. background group：0.1 μ g/ml fluorescence coupling Streptavidin is added in the reaction chamber, and room temperature places 30min, and cleaning is got rid of
It is dry；
All cleaning processes are all cleaned 3 times, every time 5 minutes using PBST.
3rd, data collection and analysis
The fluorescence signal of detection group and blank group is read using chip scanner, with fluorescence of the softwares of Genepix Pro 6.0 to acquisition
Signal is analyzed, and gathers the median in each capture antibody dots and the average value and CV values of repetition point are calculated with this.Instead
Answer and unlabelled protein sample and anti-acetylated lysine antibody added in room, it is acetylation signal that it, which scans obtained signal,
And the protein sample of biotin labeling is added in reative cell, it is albumen background signal that it, which scans obtained signal,.Detect sample
The Acetylation Level of albumen represents that the ratio is more big with the ratio of acetylation signal/background signal, illustrates the acetyl of sample protein
Change level is higher, and the difference of Acetylation Level can be by acetylation signal/background signals of relatively different samples between different samples
Ratio analyzed.
Embodiment three
TSA (Trichostatin A) handles cell model
1st, experimental principle
TSA (Trichostatin A) is a kind of reversible HDAC (histone deacetylase, histon deacetylase (HDAC)) suppressions
Preparation, by suppressing the process that albumen deacetylate is rolled into a ball, improves the Acetylation Level in cell.Distinguished with DMSO and TSA
Sample is handled, DMSO is processed as check sample, contrast and pass through after being handled with protein chip and the kit detection of the present invention through TSA
The change of Acetylation Level in cell after DMSO processing, to illustrate the Detection results of the present invention.
2nd, sample preparation
Gastric cancer cell MKN45 is trained in the culture mediums of RAPI 1640 containing 10%FBS (fetal calf serum, hyclone)
Support to 70% density, cell protein is collected after being handled 18 hours with DMSO and 3 μM of TSA respectively.
3rd, test method
A, take 106The MKN45 cells handled through DMSO and TSA, add 100 μ l protein lysates, 4 DEG C of cracking 30min,
Supernatant is collected after 10000g centrifugations 15min, concentration is determined, takes wherein 100 μ g albumen to carry out biotin labeling respectively, for examining
Albumen background signal is surveyed, remaining is used to detect acetylation signal；
B, take out protein chip and close 1h with 1%BSA room temperatures, the DMSO treatment groups and TSA treatment groups of equivalent are taken respectively
Detection sample and background sample are added in two reative cells of difference of chip, are cleaned after carrying out hybridization incubation 1.5 hours at 37 DEG C；
C, in detection group reative cell, continuously add anti-acetylated lysine antibody, 37 DEG C are incubated 1 hour, PBST cleanings,
The detection secondary antibody of HRP marks is added, is incubated at room temperature 1 hour, after PBST cleanings, addition contains 0.0015%H2O2Signal expands
The tyrasamine for increasing buffer solution and biotin labeling is reacted at room temperature 10 minutes, finally adds 0.1 μ g/ml fluorescence coupling Streptavidin,
Room temperature places 30min, cleans, and dries；0.1 μ g/ml fluorescence coupling Streptavidin, room temperature are added in background group reative cell
30min is placed, is cleaned, is dried；
D, using chip scanner DMSO treatment groups and TSA treatment group sample fluorescence signals are read, with Genepix Pro 6.0
Software is analyzed it, compares acetylation signal/background signal ratio between DMSO processing and two groups of samples of TSA treatment groups
Difference.
4th, experimental result
MKN45 cells pass through TSA processing, by WB it can be found that Acetylation Level is significantly improved (Fig. 2 a).We with
The cell of this TSA inductions is model, and the second between check sample (Control groups) and TSA processing samples is detected with the present invention
Acylated difference, GATA1 and Histone (histone) albumen Acetylation Level are found by comparing relatively obvious rising,
Raise respectively 1.6 times and 3.1 times (Fig. 2 b, 2c).Experimental result is consistent with expection, illustrates protein chip and the examination of the present invention
Agent box may be advantageously employed in detection protein acetylation modification level.
Embodiment 4
P300 overexpressing cell model inspections
1st, experimental principle
P300 is a kind of acetyltransferase (HAT), and its substrate includes nearly all histone and substantial amounts of nonhistones,
The change of cellular acetylation level can be detected from other side by being overexpressed P300, so as to be verified to protein chip.
2nd, sample preparation
Stomach cancer cell AGS is cultivated to 70% density in the culture mediums of RAPI 1640 containing 10%FBS, is used
Lipofectamine2000 distinguishes GFP-transfected (Green Fluorescent Protein, green fluorescent protein) and P300 plasmids,
Transfection collects cell RNA and protein respectively after 48 hours.RNA is after reverse transcription to detect that cell is overexpressed P300 water
Flat, albumen is used to and hybridization check Acetylation Level of the present invention.
3rd, test method
A, RNA are extracted, reverse transcription is with quantifying：
10 are taken respectively5Blank and the MKN45 cells for being overexpressed P300, add 1ml Trizol lysates, RNA extraction process
Operated according to Trizol specifications, reverse transcription is with fluorescent quantitation according to Takara reverse transcription reagent box and SYBR fluorescent quantitations
Kit specification is operated.
B, protein are collected, chip hybridization detection：
B1. 10 are taken5Blank and the MKN45 cells for being overexpressed P300, add 4 DEG C of lysate cracking 30min, 10000g from
Supernatant is collected after heart 15min, biotin labeling is carried out, for detecting albumen background signal, remaining is used to detect acetylation signal；
B2. protein chip is taken out, takes equivalent detection sample and background sample to be added in the reative cell of chip respectively, is entered at 37 DEG C
Row hybridization incubation is cleaned after 1.5 hours；
B3. in detection group reative cell, anti-acetylated lysine antibody is continuously added, 37 DEG C are incubated 1 hour, PBST cleanings,
The detection secondary antibody of HRP marks is added, is incubated at room temperature 1 hour, after PBST cleanings, addition contains 0.0015%H2O2Signal expands
Increase the tyrasamine of buffer solution and biotin labeling, react at room temperature 10 minutes, finally add 0.1 μ g/ml fluorescence coupling strepto- affine
Element, room temperature places 30min, cleans, and dries；0.1 μ g/ml fluorescence coupling Streptavidin is added in background group reative cell,
Room temperature places 30min, cleans, and dries；
B4. read blanc cell using chip scanner and be overexpressed the fluorescence signal of P300 cell samples, use Genepix Pro
6.0 softwares are analyzed it, are compared blanc cell and are overexpressed the difference of acetylation signal/background signal ratio between P300 cells
It is different.
4th, experimental result
Cell transfecting P300 plasmids after 48 hours P300mRNA significantly rise (Fig. 3 a), after being detected with protein chip, find
Compared with check sample (Control groups), P300 overexpressing cell models include Histone, GATA1, EKLF, Tubulin
Multiple albumen such as (tubulin) all there occurs that Acetylation Level raises (Fig. 3 b, 3c), and wherein GATA1 and Tubulin are equal
1.6 times are raised, Histone and EKLF have raised 1.3 times.Experimental result is consistent with expection, illustrates the albumen core of the present invention
Piece and kit may be advantageously employed in detection protein acetylation modification level.
In summary, the various embodiments described above and accompanying drawing are only the part preferred embodiment of the present invention, not to limit this hair
Bright protection domain, within the spirit and principles of the invention, any modification, equivalent substitution and improvements done etc. all should
Comprising within the scope of the present invention.
Claims (10)
1. a kind of protein chip, it is characterised in that the capture antibody on substrate, the capture are distributed in 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。
2. protein chip according to claim 1, it is characterised 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。
3. protein chip according to claim 1 or 2, it is characterised in that the substrate is glass slide, plastic slide
Or diaphragm.
4. protein chip according to claim 1 or 2, it is characterised in that the protein chip also include positive control and
Negative control, the positive control is the BSA and acetylation BSA of biotin labeling, and the negative control is BSA, mouse
IgG and rabbit normal IgG.
5. protein chip according to claim 4, it is characterised in that the protein chip be respectively by the capture antibody,
Positive control and negative control, which are selected, is formed on what is be made on the substrate.
6. protein chip according to claim 5, it is characterised in that the positive control, negative control and each catch
Obtain antibody and at least three repetition point processed is put on chip.
7. protein chip described in a kind of claim 1 or 2 is used for the device for preparing acetylation modification level after detection protein translation
Application.
8. a kind of kit, it is characterised in that the kit includes the protein chip described in claim 1 or 2.
9. kit according to claim 8, it is characterised in that the kit also include anti-acetylated lysine antibody,
Detection secondary antibody, the tyrasamine of biotin labeling, amplification of signal buffer solution, the 30%H of HRP marks2O2, fluorescence coupling strepto- it is affine
Element, protein lysate, NHS- biotins, protein labeling buffer solution, mark stop buffer and super filter tube.
10. a kind of detection method for detecting Acetylation Level after protein translation, it is characterised in that the detection method right to use
Profit requires the kit described in 9, and comprises the following steps：
The preparation of step 1, sample：
1. sample is detected：Cell or tissue sample is taken, protein lysate is added, is fully centrifuged after reaction, collect supernatant and go forward side by side
Row is quantitative, that is, obtains detecting sample；
2. background sample：Take part to detect sample, add protein labeling buffer solution and mix, add NHS- biotins and be marked,
Mark stop buffer end mark is added, 1xPBS is then added, is put into super filter tube, take ultrafiltrate to preserve after centrifugation,
Obtain background sample；
Step 2, protein chip hybridization：
1. first protein chip is closed with BSA, the detection sample and background sample of equivalent is then taken, at two of protein chip
Taken detection sample and background sample is separately added into reative cell, itself and protein chip are subjected to hybridization incubation, cleaning；
2. detection group：Anti- acetylated lysine antibody is continuously added in the reaction chamber, is incubated, and cleaning adds the inspection of HRP marks
Secondary antibody is surveyed, is incubated, cleaning adds the tyrasamine reaction of amplification of signal buffer solution and biotin labeling, finally adds fluorescence coupling
Streptavidin, is placed, and is cleaned, and is dried；
3. background group：Fluorescence coupling Streptavidin is added in the reaction chamber, is placed, is cleaned, dries；
Step 3, data collection and analysis：
The fluorescence signal of detection group and background group is read using chip scanner, the fluorescence signal of acquisition is analyzed with software,
Gather the median in each capture antibody dots and the average value and CV values of repetition point are calculated with this；Add and do not mark in reative cell
The protein sample of note and anti-acetylated lysine antibody, it is acetylation signal that it, which scans obtained signal, and in reative cell plus
Enter the protein sample of biotin labeling, it is albumen background signal that it, which scans obtained signal,；Detect the acetylation of sample protein
Level is represented with the ratio of acetylation signal/background signal.
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