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  • 細胞凋亡基因ChIP qPCR芯片 Apoptosis EpiTect ChIP qPCR Array

    科技服務 > 生物芯片實驗服務 > 細胞凋亡基因ChIP qPCR芯片 Apoptosis EpiTect ChIP qPCR Array

    細胞凋亡基因ChIP qPCR芯片 Apoptosis EpiTect ChIP qPCR Array

    細胞凋亡基因ChIP qPCR芯片 Apoptosis EpiTect ChIP qPCR Array
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    Apoptosis EpiTect ChIP qPCR Array  細胞凋亡基因ChIP qPCR芯片
     Apoptosis EpiTect ChIP qPCR Array  細胞凋亡基因ChIP qPCR芯片
     
    ProductSpeciesTechnologyCat. No.
    Apoptosis EpiTect ChIP qPCR ArrayHumanHistone ModificationsGH-012A
    Apoptosis EpiTect ChIP qPCR ArrayMouseHistone ModificationsGM-012A
     
     Apoptosis EpiTect ChIP qPCR Array profiles the histone modification status or “histone code” of 84 key genes involved in programmed cell death. Histone modifications regulate chromatin structure and correlate with the transcriptional activity of associated genes. Apoptosis plays a critical role in normal biological processes requiring cell removal including differentiation, development, and homeostasis. Stress responses (such as heat shock, ischemia, unfolded proteins, and viral infection) cause badly damaged cells to undergo apoptosis. In cell culture, growth factor withdrawal and many known experimental compounds have a similar effect. An acquired defect in apoptosis activation often leads to uncontrolled cell growth, oncogenesis, and cancer. Ligand-bound tumor necrosis factor (TNF) receptors initiate apoptosis by recruiting FADD and other death domain adaptor proteins that then recruit and activate caspases. Environmental stresses trigger BCL2 protein oligomerization and insertion into the mitochondrial membrane, releasing APAF1 and other CARD family members that also oligomerize to recruit and activate caspases. Caspases promote a proteolysis cascade that degrades cellular protein targets, while the IAP protein family directly inhibits caspases. This array includes TNF ligands and their receptors, members of the BCL-2, caspase, IAP, TRAF, CARD, death domain, death effector domain, and CIDE families, as well as genes involved in the p53 and DNA damage pathways. Monitoring the histone modifications of these genes can help determine the epigenetic mechanisms controlling programmed cell death and the propensity of a cell type to undergo apoptosis normally. Using chromatin immunoprecipitation and EpiTect ChIP qPCR Arrays, research studies can easily and reliably analyze the histone modification patterns associated with a focused gene panel related to apoptosis.
    The EpiTect ChIP qPCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease
    細胞凋亡EpiTect ChIP qPCR芯片檢測參與細胞程序性死亡的84個關鍵基因的組蛋白修飾狀態或“組蛋白密碼”。組蛋白修飾調節染色質結構和與之相關基因的轉錄活性。凋亡細胞在正常生物過程中起著至關重要的作用,包括分化、發育和內穩態的調節。應激反應(如熱休克、缺血,展開的蛋白質,和病毒感染)引起嚴重受損的細胞發生凋亡。在細胞培養中生長因子消退和許多已知的實驗化合物也有相似的效果。后天的細胞凋亡激活缺陷往往導致不可控的細胞生長,腫瘤形成,和癌癥。配體腫瘤壞死因子(TNF)受體啟動細胞凋亡通過招募FADD和其他死亡結構域接頭蛋白,然后招募和激活凋亡蛋白酶。環境壓力引發BCL2蛋白質的寡聚化和插入到線粒體膜,釋放APAF1和其他寡聚化的CARD家族成員,來招募和激活凋亡蛋白酶。凋亡蛋白酶促進蛋白質級聯水解,來降解細胞蛋白質,而IAP蛋白質家族直接抑制凋亡蛋白酶。這個芯片包括腫瘤壞死因子配體及其受體、BCL - 2、caspase,IAP,TRAF,CARD,死亡域,死亡效應領域,和CIDE家族以及p53基因和DNA損傷途徑的基因。檢測這些基因的組蛋白修飾可以幫助確定表觀遺傳機制控制程序性細胞死亡和經歷正常細胞凋亡時細胞類型的傾向。使用染色質免疫沉淀反應和EpiTect ChIP qPCR芯片,可以很容易地、可靠地分析與細胞凋亡有關的基因的組蛋白修飾模式。
    TNF Ligand Family:CD40LG (TNFSF5), CD70 (TNFSF7), FASLG (TNFSF6), LTA (TNFB), TNF, TNFSF10 (TRAIL), TNFSF8.
    TNF Receptor Family:CD27 (TNFRSF7), CD40 (TNFRSF5), FAS (TNFRSF6), LTBR, TNFRSF10A, TNFRSF10B (DR5), TNFRSF11B, TNFRSF1A, TNFRSF21, TNFRSF25 (DR3), TNFRSF9.
    BCL-2 Family: BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL2, BCL2A1 (Bfl-1/A1), BCL2L1 (BCL-X), BCL2L10, BCL2L11, BCL2L2, BCLAF1, BID, BIK, BNIP1, BNIP2, BNIP3, BNIP3L, HRK, MCL1.
    Caspase Family:CASP1 (ICE), CASP10 (MCH4), CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8 (FLICE), CASP9.
    IAP Family:BIRC2 (c-IAP2), BIRC3 (c-IAP1), BIRC6, BIRC8, XIAP.
    TRAF Family:TRAF2, TRAF3, TRAF4.
    CARD Family:APAF1, BCL10, BIRC2, BIRC3, NOD1 (CARD4), CARD6, CARD8, CASP1 (ICE), CASP2, CASP4, CASP5, CASP9, CRADD, NOL3, PYCARD (TMS1/ASC), RIPK2.
    Death Domain Family:CRADD, DAPK1, FADD, FAS (TNFRSF6), TNFRSF10A, TNFRSF10B (DR5), TNFRSF11B, TNFRSF1A, TNFRSF21, TNFRSF25 (DR3), TRADD.
    Death Effector Domain Family:CASP8 (FLICE), CASP10 (MCH4), CFLAR (CASPER), FADD.
    CIDE Domain Family: CIDEA, CIDEB, DFFA.
    p53 and DNA Damage Response:ABL1, AKT1, APAF1, BAD, BAX, BCL2, BCL2L1 (BCL-X), BID, CASP3, CASP6, CASP7, CASP9, GADD45A, TP53, TP53BP2, TP73.
    Anti-Apoptosis: AKT1, BAG1, BAG3, BCL2, BCL2A1 (Bfl-1/A1), BCL2L1 (BCL-X), BCL2L10, BCL2L2, BFAR, BIRC2 (c-IAP2), BIRC3 (c-IAP1), BIRC6, BIRC8, BNIP1, BNIP2, BNIP3, BRAF, CASP2, CD27 (TNFRSF7), CFLAR (CASPER), FAS (TNFRSF6), IGF1R, MCL1, NFKB1, TNF, XIAP.

    實驗原理與流程

    The ChIP PCR array is a set of optimized real-time PCR primer assays on 96-well or 384-well plates for pathway or disease focused analysis of in vivo protein-DNA interactions. The ChIP PCR array performs ChIP DNA analysis with real-time PCR sensitivity and the multi-genomic loci profiling capability of a ChIP-on-chip. Simply mix your ChIP DNA samples with the appropriate ready-to-use PCR master mix, aliquot equal volumes to each well of the same plate, and then run the real-time PCR cycling program. (Download user manual)

    What ChIP PCR Array Offers?

    • Function or Disease Focused: Arrays represent a panel of genomic regions relevant to a biological function or disease state.

    • Reliable & Sensitive: Arrays can analyze multiple genomic regions simultaneously with Real-Time PCR precision and sensitivity.

    • Easy to Use Data Analysis: Download an easy-to-use Excel-based data analysis template [here]. Data analysis is based on the ΔΔCt method with normalization of the specific antibody and control IgG raw data to input raw data.

    Layout and Controls: The PCR Arrays are available in both 96- and 384-well plates and are used to monitor the expression of 84 genes related to a disease state or pathway plus five housekeeping genes. Controls are also included on each array for ChIP DNA quality controls and general PCR performance.
    You can easily perform a ChIP PCR Array experiment in your own laboratory, or send your samples to us and take advantage of our 
    PCR Array Services.

     

    Performance
    EpiTect Chip qPCR Arrays provide the high sensitivity, specificity and reproducibility using SYBR-based real-time PCR technology.

    Sensitivity
    Together with our easy and fast One-Day ChIP kit, ChIP-Grade Antibody Kits, one million cells per assay as starting material provides 100% effective call rates.

    Ct Range

     

    Percent Distribution of Ct Values
    InputH3K4me3Control IgG
    <240%27%0%
    25-30100%60%0%
    30-350%13%96%
    Absent Calls0%0%4%

    Table 1. ChIP PCR Arrays Analyze the Enrichment of 84 Genomic Sites with as Little as One Million Cells. P19 mouse embryonic carcinoma cells were prepared for ChIP Assay using the EpiTect Chip One-Day Kit and anti-H3K4me3 Antibody Kit. One million cells were used as starting material for each ChIP Assay. The purified ChIP DNA samples were characterized using Mouse Stem Cell Transcription Factor ChIP PCR Array with 1/100th of the ChIP DNA as template in each well. The Real-Time PCR results demonstrate 100 % effective call rates for the Input Fraction (Ct < 30). The difference of Ct value between the anti-H3K4me3 antibody and the control IgG fractions indicates the specific enrichment of the antibody, whereas the high Ct value of the control IgG fraction indicates the low background of the assay.

    Reproducibility
    The complete ChIP PCR Array System demonstrates a high degree of reproducibility across technical replicates, lots, instruments, and different handling, insuring reliable detection of differences in genomic DNA enrichment among biological samples.

    Figure 5. Consistent Performance within the Same Plate or across Different Plates. Sonicated chromatin from HeLa cells (20 μg) was immunoprecipitated with 2 μg of anti-H3ac antibody or control IgG for 2 hours using the EpiTect Chip One-Day Kit. The obtained ChIP DNA samples were characterized in triplicates with EpiTect Chip qPCR primers specific for the active genes (GAPDH, RPL30, ALDOA), inactive genes (MYOD1, SERPINA), repetitive sequence (SAT2, SATa), and an ORF-free region (IGX1A) either within the same array plate or among different array plates in order to evaluate the intra- and inter-plate consistency. The anti-H3ac antibody enriched genomic DNA at active gene promoter regions with a high signal-to-noise ratio and a low co-efficiency of variation (less than 2.02%), irrespective of the type of assay (intra or inter-plate)

    Figure 6. Consistent Performance with Various Amount of DNA Samples, Instruments or Handling Conditions. All experiments were performed in triplicates. Cells from MCF-7 (1 million per sample) were subjected to ChIP assay with anti-RNA Polymerase II (Pol 2) antibody followed by qPCR analysis of the proximal promoter of GAPDH, and an ORF-free region (IGX1A). Researcher A & B performed the PCR assays either in 96-well plate or 384-well plate format, on a Stratagene MX 3005 or an ABI 7900 Real-Time PCR instrument respectively. The same ChIP DNA samples were used which were stored for extended periods of time as indicated. The results demonstrate high reproducibility of PCR performance across technical replicates, lots, instruments, and differential handling.

    Specific and Accurate ChIP-qPCR Detection
    One prerequisite for ChIP PCR Array technology is the uniform and high
    PCR amplification efficiency allowing a reciprocal comparison of ChIP enrichment among all genomic loci analyzed. The unique combination of SABiosciences' proprietary ChIP-qPCR primer design algorithm, rigorous validation of every ChIP-qPCR primer assay, and high performance SYBR Green master mix guarantees superior performance of EpiTect Chip qPCR Arrays.

    A:

    B:

    Figure 7. Uniform Amplification Efficiency and Specific PCR Detection. 96 ChIP-qPCR primers were randomly picked from our genome-wide primer pool and analyzed for their performance. (A) All assays exhibit an average amplification efficiency of 99% with a 104.5% confidence interval between 102.5-105.2%, the uniform high amplification efficiency ensures accurate analysis of multiple genomic loci simultaneously using ΔΔCt method. (B) Each ChIP-qPCR primer assay is experimentally validated using dissociation (melt) curve analysis and agarose gel verification. Each pair of primers on PCR Array produces a single specific product as indicated by a single Dissociation Curve peak at a melting temperature (Tm) greater than 75 oC, and PCR product was further validated on agarose gel for a single product of the predicted size without secondary products such as primer dimers

    Application Examples

    EpiTect Chip qPCR Arrays provide streamlined approaches to 1) Study biology or disease-focused gene regulation through histone modification and transcriptional regulatory network; 2) Monitor the dynamics of chromatin structure in the screening of function-specific epigenetic patterns; 3) Validate ChIP-on-chip or ChIP-seq results. The EpiTect Chip qPCR Arrays are also powerful tools for studying the mechanism contributing to gene expression changes observed by RT2 Profiler PCR Arrays.

    Below are listed a few examples of application data generated by our R&D group. To see the research using ChIP PCR Arrays published by the scientific community, please see our Publication List:http://www.sabiosciences.com/support_publication.php

    Stem Cell Research

    Stem cell differentiation into specific tissues involves the complex yet coordinated action of many transcription factors regulating not only tissue-specific genes, but also genes essential for differentiation itself. Histone modifications at the promoters of transcription factors are key mechanism regulating their expression. We used EpiTect Chip qPCR Arrays and RT2 PCR Arrays to monitor the dynamic coordination of epigenetic modification and gene expression during retinoic acid (RA) induced differentiation of P19 mouse embryonic carcinoma cells (Figure 1). This RA treatment differentiates pluripotent P19 cells into somatic cells (Figure 2). The EpiTect Chip qPCR Array data showed that both gene expression and histone modifications on key transcription factors were changed in a dynamic manner through the course of P19 cell differentiation (Figure 3).

    Figure 1. Schematic Representation of Pluripotency-Associated Gene Dynamics throughout Stem Cell Differentiation

    Figure 2. Retinoic Acid (RA) Differentiation of Mouse Embryonic Carcinoma P19 Cells.

    Figure 3. Dynamic Epigenetic Alternations and Gene Expression Changes during RA-Induced P19 Differentiation. ChIP PCR Arrays and RT2 PCR Arrays were used to monitor the changes in gene expression levels and histone modification marks (H3Ac, H3K4me3, H3K27me3, and H3K9me3). The promoter region and expression levels of 84 key stem cell transcription factors were simultaneously analyzed during RA-induced neurogenesis of P19 cells at various time points (day 0, 4, and 8). Primer sets for the +1kb region downstream of the transcription start sites of the 84 genes and 12 control regions were preloaded on the ChIP PCR Array. Cluster analysis (http://www.sabiosciences.com/chippcrarray_data_analysis.php) of histone marks and mRNA level changes for the 84 genes were visualized as a heat map to represent the fold-differences during the RA-induced differentiation at the specified time points.

    Characterize the Pattern of Histone Modifications

    EpiTect Chip qPCR Arrays can be used to monitoring differential histone modifications across a gene.

    Figure 4. The Custom EpiTect Chip 30Kb Tiling Array Quickly Maps Histone Modifications Surrounding the Transcription Start Site (TSS) of CDKN1A Gene. EpiTect Chip Antibodies against modified histones (H3Ac, H3K4me2, H3K27me3), or NIS were used to precipitate chromatin from one million HeLa cells. Each ChIP DNA fraction was analyzed with Custom EpiTect Chip 30Kb Tiling Array representing 30 one-kb tile intervals across the promoter region of the CDKN1A gene. The results indicate the enrichment of histone markers for actively transcribed genes (H3Ac and H3K4me2) but not marks for transcriptional inactive genes (H3K27me3) in the genomic region surrounding the TSS of CDNK1A.


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