CN205539001U - Detect protein acetylation horizontally albumen chip and kit - Google Patents

Abstract

The utility model discloses a detect protein acetylation horizontally albumen chip and kit can detect protein translation back acetylation level, the albumen chip includes substrate and the array antibody of catching on the substrate that distributes, the substrate has the glass substrate of the three -dimensional material of agarose membrane for the peridium, and the proteopexy of this substrate is efficient, and the stable performance, it includes 68 kind at least specific antibody to catch antibody. The kit contains the albumen chip, still includes collection of anti acetylation signal detect reagent and albumen and mark reagent. Use the utility model discloses an albumen chip and kit, the proteopexy is efficient, and the stable performance to the back acetylation level of decorateing is translated to a plurality of protein among the sample detecting simultaneously.

Description

The protein chip of detection protein Acetylation Level and test kit

Technical field

This utility model relates to protein chip and test kit, particularly relates to one and can detect acetylation modification after protein translation The protein chip of level and comprise the test kit of this protein chip.

Background technology

Along with in scientist's discovery first to acetylation of histone such as Phillips, Allfrey sixties in last century, in the 50 of the past Nian Zhong, mankind's understanding acetylizad to protein has had significant progress.Protein acetylation modification is divided into two kinds, and one is egg During white translation, the acetylation modification of N end, all has an impact synthesis, location and the stability of albumen.Another kind is protein translation After acetylation modification on lysine residue epsilon-amino, under acetyltransferase (HAT) is catalyzed, the second of S-acetyl-coenzyme-A Acyl group group is transferred to the lysine of destination protein, this process the most also by histon deacetylase (HDAC) (HDAC) can Inverse regulation.Acetylation is the important component part 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, at Cytoplasm and mitochondrion In all there is substantial amounts of protein acetylation, participated in RNA shearing, cell cycle, DNA replication dna, transcribe, cell consideration convey In the multiple bioprocesss such as fortune.Additionally a large amount of intermediate supersession enzymes can be acetylation modifications, and its Dynamic Regulating Process is thin to control Born of the same parents' metabolism has important function.The regulation of Acetylation Level is out of control can result in multiple disease, the acetylation water of multiple transcription factor Flat generation and the development all affecting (including P53, STAT3, HIF-1 α etc.) cancer, the most existing multiple with HDAC as target The clinical medicine such as SAHA (Vorinostat) of point is used for the treatment of cancer-related diseases.Therefore, detect the most efficiently In sample, the Acetylation Level of protein is the most significant in scientific research, medical diagnosis and pharmaceutical developments field.

Although lysine acetylation has important function as a kind of common post translational modification, but due to relatively low abundance Level, the detection of Acetylation Level needs to be enriched with acetylated protein in total protein.But acetylation antibody is to acetylation The affinity of albumen is relatively low, it is impossible to meets and is directly enriched with acetylated protein, but after needing proteopepsis is become peptide fragment, Re-using antibody enrichment acetylation peptide fragment, the means through mass spectral analysis detect.It is well known that mass spectral analysis needs are right Sample carries out fragmentation process, and this preprocessing process is extremely complex, needs to take a substantial amount of time and manpower, with high costs, Mass spectral analysis simultaneously is the highest 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 a Measurement for Biotechnique getting up of developed recently.Profit By this technology, by by intensive to lot of antibodies or antigen place system on solid phase carrier, have and can detect multiple proteins simultaneously And the feature that sample requirement amount is little, the research for high flux gene expression has very important significance.But, the most still do not have The disclosure of the correlation techniques such as the protein chip and the test kit that are related to acetylation modification level after detecting protein translation.

Inventor, according to the research experience for many years in protein chip field, have developed a kind of protein chip, it is possible to be used for examining Survey acetylation modification level after protein translation, also disclose the preparation method of this protein chip, application, test kit and inspection simultaneously The correlation techniques such as survey method, the above-mentioned technology of inventor's exploitation has filled up acetylation modification level after detecting protein translation The technological gap in protein chip field.

Utility model content

One of technical problem to be solved in the utility model is, it is provided that a kind of protein chip, it is possible to simultaneously many in detection sample Planting acetylation modification level after protein translation, and proteopexy efficiency is high, detection sensitivity is high.

The two of technical problem to be solved in the utility model are, it is provided that a kind of test kit, comprise described protein chip, it is possible to Conveniently acetylation modification level after the translation of detection multiple proteins.

For solving one of above-mentioned technical problem, the protein chip that the present invention provides, resist including substrate and the capture being distributed on substrate Body, described substrate is the glass substrate being coated with agarose film three-dimensional material, and described substrate is fixed with the institute of distribution in array Stating capture antibody, described capture antibody includes but not limited to following 68 strain specific antibodieies: 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。

Described capture antibody is following 68 strain specific antibodieies: 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。

Described protein chip, also includes that positive control and negative control, described positive control are biotin labeled BSA (BSA- Biotin) and acetylation BSA (Ac-BSA), described negative control is BSA (bovine serum albumin), mouse IgG and rabbit normal IgG。

Described positive control, negative control and each capture antibody all systems at least 3 on substrate repeat a little.

Above-mentioned substrate is the glass substrate being coated with agarose film three-dimensional material, and this three-dimensional material has loose structure, Ke Yijie Closing more albumen, proteopexy efficiency is high, and stable performance, can improve the detection sensitivity of chip, can detect the dense of albumen Degree reaches pg/ml rank.Meanwhile, chip will not be examined by the chip using this three-dimensional material when using follow-up optical detection Survey result and produce interference and impact.

Above-mentioned 68 strain specific antibodieies are through well-chosen.For realizing the high efficiency acetylation detecting multiple proteins simultaneously Level, inventor to substantial amounts of it may happen that the acetylizad antibody of protein conducts in-depth research, filtered out above-mentioned 68 kinds can Occur that obvious acetylation modification, atopic are good, substantially and signal is preferable linear relationship with antigen concentration to detection signal Specific antibody, 68 strain specific antibodieies disclosed by the invention may be advantageously employed in the detection of protein acetylation modification level, Its testing result can the protein acetylation modification level of reaction detection sample on the whole.Rely on above-mentioned 68 species specificity simultaneously Antibody, can increase and put make potential protein acetylation can occurring or need to check albumen for the needs of different experiments Other different antibody whether matter acetylation modification level changes, it is possible to meet the requirement of personalized customization.

For solving the two of above-mentioned technical problem, the test kit that this utility model provides, including aforesaid protein chip.

Concrete, described test kit also includes that acetylation signal detection reagent, protein collect labelled reagent and super filter tube, described Acetylation signal detection reagent is in order to detect the acetylation signal in protein sample and protein local signal, described collecting protein Labelled reagent is in order to crack tissue or cell, and crack protein is carried out biotin labeling.

Further, described acetylation signal detection reagent includes that protein hybridization buffer, acetylated lysine antibody, HRP mark Detection two anti-, biotin labeled tyramine, amplification of signal buffer, the 30%H of note₂O₂, fluorescence coupling Streptavidin.

Further, described collecting protein labelled reagent include protein lysate, NHS-biotin, biotin labeling buffer, Labelling stop buffer.

It is as follows that protein chip of the present utility model and test kit are used for detecting protein Acetylation Level detection method:

1, the preparation of sample

1. detect sample: take cell or tissue sample, add protein lysate, fully centrifugal after reaction, collect supernatant and go forward side by side Row quantitatively, i.e. obtains detecting sample (being used for detecting acetylation signal)；

2. background sample: take part detection sample, add the mixing of protein labeling buffer, add NHS-biotin and be marked, Add labelling stop buffer end mark, be subsequently adding 1xPBS (phosphate buffered saline(PBS)), put into super filter tube, by centrifugation After take ultrafiltrate preserve, i.e. obtain background sample (being used for detecting albumen background signal).

2, protein chip hybridization

The most first protein chip BSA is closed, then take detection sample and the background sample of equivalent, at two of protein chip Reative cell is separately added into taken detection sample and background sample, itself and protein chip are carried out hybridization incubation, clean；

2. detection group: continuously add anti-acetylated lysine antibody in the reaction chamber, hatch, cleans, and adds the inspection of HRP labelling Survey two to resist, hatch, clean, add amplification of signal buffer and the reaction of biotin labeled tyramine, finally add fluorescence coupling Streptavidin, places, and cleans, and dries；

3. background group: add fluorescence coupling Streptavidin in the reaction chamber, place, clean, dry.

3, data collection and analysis

Chip scanner is used to read detection group and the fluorescence signal of background group, with Genepix Pro 6.0 software fluorescence to obtaining Signal is analyzed, and gathers the fluorescence signal median in each capture antibody dots and calculates the meansigma methods repeated a little and CV with this Value.Adding unlabelled protein sample and anti-acetylated lysine antibody in reative cell, the signal that its scanning obtains is acetyl Changing signal, and add biotin labeled protein sample in reative cell, the signal that its scanning obtains is albumen background signal. The ratio of the Acetylation Level acetylation signal/background signal of sample protein represents, this ratio the biggest then explanation sample protein Acetylation Level is the highest.

Between sample, the difference of Acetylation Level is analyzed according to the ratio of acetylation signal/background signal.

This utility model provides first and a kind of can detect the protein chip of multiple protein acetylation modification levels in sample simultaneously And test kit, compared to the method using mass spectral analysis detection protein Acetylation Level in prior art, the present invention is without to egg White matter carries out the preprocessing process such as enzyme action purification of complexity, can solve to detect at present that protein acetylation modification wastes time and energy asks Topic, and the multiple proteins in sample can be detected simultaneously, detection is more comprehensive, and detection efficiency is high, and sample requirements is little, Research for high flux gene expression has very important significance, and relies on this platform simultaneously, can be for the need of different experiments The antibody that main points system is different, it is possible to meet the requirement of personalized customization.

Accompanying drawing explanation

Fig. 1 is the structural representation of protein chip one embodiment of the present utility model.

Fig. 2 is the operational flowchart of protein chip of the present utility model and test kit.

Fig. 3 is the part detection knot of the acetylation change of protein chip of the present utility model and test kit detection TSA inducing cell Really.

Fig. 4 is the part detection knot of the acetylation change of protein chip of the present utility model and test kit detection P300 overexpressing cell Really.

Detailed description of the invention

Below in conjunction with experimental data and accompanying drawing, the technical solution of the utility model is carried out clear, complete description, it is clear that institute The embodiment described is a part of embodiment of the present utility model rather than whole embodiments.Based in this utility model Embodiment, the every other embodiment that those of ordinary skill in the art are obtained on the premise of not making creative work, Broadly fall into the scope of this utility model protection.

Embodiment one

A kind of protein chip detecting protein Acetylation Level that this utility model provides, including substrate and being distributed on substrate Capture antibody, described substrate is the glass substrate being coated with agarose film three-dimensional material, described substrate is fixed with and divides in array The described capture antibody of cloth, described capture antibody includes but not limited to following 68 strain specific antibodieies: 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 of the protein chip of the present embodiment is the glass substrate being coated with agarose film three-dimensional material, the detection effect of protein chip Can depend on the stability of protein and bearer plane, on traditional chip carrier, protein fixed efficiency is low, and unstable, this Limit the application of protein chip.Agarose film three-dimensional material has the loose structure of three-dimensional, this three dimensional structure and protein Binding ability is strong and combines stable, and proteopexy efficiency is high, is coated with the protein chip of the glass substrate of agarose film three-dimensional material, The detection sensitivity of chip in conjunction with the most monolateral, can be improve, the concentration as little as pg/ml rank of albumen can be detected.And, This chemical unit material will not produce interference and impact to protein chip when follow-up optical detection on chip detection result.

The protein chip of the present embodiment is rectangular structure, this protein chip is provided with multiple reative cell, sets in each reative cell Having multiple loading wells (or point of sample), loading wells is matrix form distribution, and each loading wells all puts above-mentioned 68 strain specific antibodieies of system In one, every strain specific antibodies is put successively and is formed in three continuous print loading wells, as it is shown in figure 1, be provided with on this substrate 1 × 4 totally 4 reative cells, are provided with the loading wells that 20 row × 12 row are matrix distribution in each reative cell, wherein the first row from Left-to-right positive control A1, A2 the most processed and 01 to No. 18 specific antibody AML1, AMPK, APE, AR, α-Tubulin, β-Catenin, Bcl-6, Beclin, BRAF, MYB, c-Myc, CREB, CTBP2, DEK, E2F1, E2F2, E2F3, EGFR, the second row and the third line repeat the point sample mode of the first row, and the most each specific antibody repeats point sample 3 times.Depend on Secondary analogize, use same point sample mode successively by 19 to No. 38 specific antibodies Her2, Her4, ER, EKLF, FEN1, FOXO1, FOXO3, FOXO4, GATA1, GATA2, GATA3, GATA4, GR, HIF-1 α, HMGA1, HMGB1, HSP90, IRF2, IRS1, KRAS point is formed on the 4th to 6 row, successively by 39 to No. 58 specific antibodies Ku70, and MDM2, MEF2A,NEIL2,NFκB,Notch1,P21,P53,P65,PGC1,PTEN,RB1,KPNA2,RUNX2,SMAD2, SMAD4, SMAD7, SOX4, SOX9, SREBP1 point is formed on the 7th to 9 row, successively by 59 to No. 68 specific antibodies SRY, STAT1, STAT2, STAT3, TDG, VEGF, WRN, β-Actin, GAPDH, BSA point is formed on the 10th to 12 row Front 10 row.

In the preferred implementation of the present embodiment, it is raw that the 19th and 20 row of the 10th to 12 row may be used for a positive control processed The BSA (BSA-biotin) of thing element labelling and acetylation BSA (Ac-BSA), the 10th walk to the 16th of 12 row the, 17, 18 row may be used for a negative control BSA (bovine serum albumin) processed, mouse IgG and rabbit normal IgG.

In the more preferably embodiment of the present embodiment, the 10th the 11st to 15 row walking to 12 row are also used as expansion module, Other antibody of special requirement point system according to user, it is possible to meet the requirement of personalized customization.Point on the substrate of the present embodiment Sample hole can be to be arranged to other matrix distribution modes arbitrarily.

Embodiment two

The preparation method of protein chip is as follows:

68 strain specific antibodies PBS-15% glycerol are diluted to 0.5 μ g/ μ l, use chip point platform that antibody is put system one by one On substrate, substrate is the glass substrate being coated with agarose film three-dimensional material.Each spot diameter 200 μm, point and dot spacing 500μm；Use biotin labeled BSA (BSA-biotin), acetylation BSA (Ac-BSA) as positive control, make With BSA, mouse IgG, rabbit normal IgG as negative control；Sun ginseng, cloudy ginseng and capture antibody all put 3 weights of system Complex point, institute, a little in matrix distribution, is prepared as protein chip.Described 68 strain specific antibodieies are: 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。

In above-mentioned 68, specific antibody is through well-chosen, may be advantageously employed in the inspection of protein acetylation modification level Surveying, its testing result can the protein acetylation modification level of reaction detection sample on the whole.Otherwise for different experiments Purpose, relies on the protein chip of embodiment one, the antibody that some fixture is the most different, and is used for detecting the protein of sample Acetylation Level, it is possible to meet the requirement of personalized customization.

Embodiment three

The detection method (as shown in Figure 2) of protein chip of the present utility model and test kit:

1, the preparation of sample

1. sample is detected: take 10⁶–5x 10⁶Cell or 10 40mg tissue samples, add 100 200 μ l protein lysates, Placing 30 minutes at 4 DEG C and frequently vibrate, then at 4 DEG C, 10000g is centrifuged 15 minutes, collects supernatant and carries out determining Amount, i.e. obtains detecting sample (being used for detecting acetylation signal)；

2. background sample: take 100 μ g and detect sample, adds protein labeling buffer and mixes to 50 μ l, add 2 μ l NHS-biological Element at room temperature reacts 2 hours and is marked, and adds 1.6 μ l labelling stop buffers in 30 minutes end marks of room temperature reaction, It is subsequently adding 450 μ l 1xPBS, puts into super filter tube, after 4 DEG C of 10000g are centrifuged 30 minutes, take ultrafiltrate in-20 DEG C of preservations, I.e. obtain blank sample (being used for detecting albumen background signal).

2, protein chip hybridization

The most first protein chip 1%BSA room temperature is closed 1h, then take detection sample and the blank sample of equivalent, at albumen Two reative cells of chip are separately added into taken detection sample and blank sample, it is carried out miscellaneous with protein chip at 37 DEG C Friendship is cleaned after hatching 1.5 hours；

2. detection group: continuously add anti-acetylated lysine antibody in the reaction chamber, hatch 1 hour for 37 DEG C, PBST cleans, adds The detection two entering HRP labelling resists, and incubated at room 1 hour, after PBST cleans, adds containing 0.0015%H₂O₂Amplification of signal Buffer and biotin labeled tyramine room temperature reaction 10 minutes, finally add 0.1 μ g/ml fluorescence coupling Streptavidin, Room temperature places 30min, cleans, and dries；

3. background group: add 0.1 μ g/ml fluorescence coupling Streptavidin in the reaction chamber, room temperature places 30min, cleans, gets rid of Dry；

All cleaning processes all use PBST clean 3 times, each 5 minutes.Two reative cells carry out background detection the most respectively And pattern detection, it is ensured that pattern detection and background detection carry out under similarity condition, improve result of the test effectiveness and can Comparative.

3, data collection and analysis

Chip scanner is used to read detection group and the fluorescence signal of blank group, with Genepix Pro 6.0 software fluorescence to obtaining Signal is analyzed, and gathers the median in each capture antibody dots and calculates the meansigma methods repeated a little and CV value with this.Instead Answering and add unlabelled protein sample and anti-acetylated lysine antibody in room, the signal that its scanning obtains is acetylation signal, And reative cell adds biotin labeled protein sample, the signal that its scanning obtains is albumen background signal.Detection sample The ratio of the Acetylation Level acetylation signal/background signal of albumen represents, this ratio is the biggest, and the acetyl of sample protein is described Change level is the highest, and between different samples, the difference of Acetylation Level can be by the acetylation signal/background signal of relatively different samples Ratio analyzed.

Embodiment four

TSA (Trichostatin A) processes cell model

1, experimental principle

TSA (Trichostatin A) is that a kind of reversible HDAC (histone deacetylase, histon deacetylase (HDAC)) presses down Preparation, the process rolled into a ball by suppression albumen deacetylate, improves the Acetylation Level in cell.With DMSO and TSA respectively Processing sample, DMSO is processed as check sample, and protein chip and test kit detection by the present invention contrast warp after TSA processes The change of Acetylation Level in cell after DMSO process, to illustrate the Detection results of the present invention.

2, prepared by sample

Gastric cancer cell MKN45 is trained in RAPI 1640 culture medium containing 10%FBS (fetal calf serum, hyclone) Support the density to 70%, after processing 18 hours with DMSO and 3 μMs of TSA respectively, collect cell protein.

3, test method

A, take 10⁶Through the MKN45 cell that DMSO and TSA processes, add 100 μ l protein lysate 4 DEG C cracking 30min, 10000g collects supernatant after being centrifuged 15min, measures concentration, takes wherein 100 μ g albumen respectively and carry out biotin labeling, be used for examining Surveying albumen background signal, remaining is used for detecting acetylation signal；

B, take out protein chip 1%BSA room temperature and close 1h, take the DMSO process group of equivalent and TSA process group respectively Detection sample and background sample join in two reative cells of difference of chip, clean after carrying out hybridization incubation 1.5 hours at 37 DEG C；

C, in detection group reative cell, continuously add anti-acetylated lysine antibody, hatch 1 hour for 37 DEG C, PBST clean, The detection two adding HRP labelling resists, and incubated at room 1 hour, after PBST cleans, adds containing 0.0015%H₂O₂Signal expands Increase buffer and biotin labeled tyramine room temperature reaction 10 minutes, finally add 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 is added in background group reative cell Place 30min, clean, dry；

D, use chip scanner read DMSO process group and TSA process group sample fluorescence signal, with Genepix Pro 6.0 It is analyzed by software, compares DMSO and processes acetylation signal/background signal ratio between two groups of samples of TSA process group Difference.

4, experimental result

MKN45 cell is through TSA process, by WB it appeared that Acetylation Level significantly improves (a in Fig. 3).I The cell induced with this TSA as model, detect check sample (Control group) by the present invention and TSA process between sample Acetylation difference, by comparison find GATA1 with Histone (histone) albumen Acetylation Level have relative obvious Rise, raised 1.6 times and 3.1 times (b, c in Fig. 3) respectively.Experimental result is consistent with expection, and the egg of the present invention is described White chip and test kit may be advantageously employed in detection protein acetylation modification level.

Embodiment five

P300 overexpressing cell model inspection

1, 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 by process LAN P300 in terms of another, thus protein chip is verified.

2, prepared by sample

Stomach cancer cell AGS cultivates the density to 70% in RAPI 1640 culture medium containing 10%FBS, uses Lipofectamine2000 GFP-transfected (Green Fluorescent Protein, green fluorescent protein) and P300 plasmid respectively, Cell RNA and protein is collected respectively after transfecting 48 hours.RNA after reverse transcription in order to detect cell process LAN P300 water Flat, albumen in order to hybridization check Acetylation Level of the present invention.

3, test method

A, RNA extraction, reverse transcription are with quantitative:

Take 10 respectively⁵The blank MKN45 cell with process LAN P300, adds 1ml Trizol lysate, and RNA extracts process Operating according to Trizol description, reverse transcription and fluorescent quantitation are according to Takara Reverse Transcription box and SYBR fluorescent quantitation Test kit description operates.

B, protein are collected, chip hybridization detection:

B1. 10 are taken⁵Blank and the MKN45 cell of process LAN P300, add lysate 4 DEG C cracking 30min, 10000g from Collecting supernatant after heart 15min, carry out biotin labeling, be used for detecting albumen background signal, remaining is used for detecting acetylation signal；

B2. take out protein chip, take equivalent detection sample respectively and background sample joins in the reative cell of chip, enter at 37 DEG C Row hybridization incubation cleaned after 1.5 hours；

B3. in detection group reative cell, continuously adding anti-acetylated lysine antibody, hatch 1 hour for 37 DEG C, PBST cleans, The detection two adding HRP labelling resists, and incubated at room 1 hour, after PBST cleans, adds containing 0.0015%H₂O₂Signal expands Increase buffer and biotin labeled tyramine, room temperature reaction 10 minutes, finally add 0.1 μ g/ml fluorescence coupling strepto-affine Element, room temperature is placed 30min, is cleaned, dries；0.1 μ g/ml fluorescence coupling Streptavidin is added in background group reative cell, Room temperature places 30min, cleans, and dries；

B4. use chip scanner to read blanc cell and the fluorescence signal of process LAN P300 cell sample, use Genepix Pro It is analyzed by 6.0 softwares, compares the difference of acetylation signal/background signal ratio between blanc cell and process LAN P300 cell Different.

4, experimental result

Cell transfecting P300 plasmid after 48 hours P300mRNA significantly rise (a in Fig. 4), with protein chip detect after, Find compared with check sample (Control group), P300 overexpressing cell model include Histone, GATA1, EKLF, Multiple albumen such as Tubulin (tubulin) all there occurs that Acetylation Level raises (b, c in Fig. 4), wherein GATA1 All raised 1.6 times with Tubulin, Histone and EKLF has raised 1.3 times.Experimental result is consistent with expection, and this is described Bright protein chip and test kit may be advantageously employed in detection protein acetylation modification level.

In sum, the various embodiments described above and accompanying drawing are only part preferred embodiment of the present utility model, not in order to limit Protection domain of the present utility model, all within spirit of the present utility model and principle, any amendment of being made, equivalent, Improve, all should be included in protection domain of the present utility model.

Claims (8)

1. a protein chip, it is characterised in that including substrate and the capture antibody being distributed on substrate, described substrate is for being coated Have the glass substrate of agarose film three-dimensional material, described substrate be fixed with in array the described capture antibody of distribution, described in catch Obtain antibody and include at least following 68 strain specific antibodieies: 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。

Protein chip the most according to claim 1, it is characterised in that described capture antibody is following 68 strain specific antibodieies: 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。

Protein chip the most according to claim 1 and 2, it is characterised in that described protein chip also include positive control and Negative control, described positive control is biotin labeled BSA and acetylation BSA, and described negative control is BSA, mouse IgG and rabbit normal IgG.

Protein chip the most according to claim 3, it is characterised in that described positive control, negative control and each catch Obtain antibody all systems at least 3 on substrate to repeat a little.

5. a test kit, it is characterised in that described test kit includes the protein chip described in claim 1 or 2.

Test kit the most according to claim 5, it is characterised in that described test kit also include acetylation signal detection reagent, Protein collects labelled reagent and super filter tube, and described acetylation signal detection reagent is in order to detect the acetylation letter in protein sample Number and protein local signal, crack protein, in order to crack tissue or cell, and is entered by described collecting protein labelled reagent Row biotin labeling.

Test kit the most according to claim 6, it is characterised in that described acetylation signal detection reagent includes protein hybridization Buffer, acetylated lysine antibody, HRP labelling detection two is anti-, biotin labeled tyramine, amplification of signal buffer, 30%H₂O₂, fluorescence coupling Streptavidin.

Test kit the most according to claim 6, it is characterised in that described collecting protein labelled reagent include protein lysate, NHS-biotin, biotin labeling buffer, labelling stop buffer.