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DYKDDDDK Tag CUTANA™ CUT&RUN Antibody介绍

更新时间:2024-03-13   点击次数:133次

DYKDDDDK抗体可用于FLAG®*标记的靶蛋白研究。该抗体符合EpiCypher“CUTANA Compatible"标准,适用于利用核酸酶靶向切割和释放(CUT & RUN)和靶向剪切及转座酶技术(CUT & Tag)进行基因组定位的方法。每个批次的CUTANA Compatible抗体均使用EpiCypher优化的方案,按照指-定的方法进行测试,并确定产峰显示的基因组分布模式与所报告的靶标功能一致。DYKDDDDK Tag 抗体在表达 3xFLAG 标记的 GATA3 转录因子的乳腺癌细胞中,产生与 GATA3 重叠的 CUT&RUN 峰(图 2-3[1]**


*FLAG® is a registered trademark of Merck KGaA, Darmstadt, Germany.

**Thanks to Dr. Takaku (UND) for 3xFLAG-GATA3-3xHA MDA-MB-231 cells.


产品详情

产品名称:DYKDDDDK Tag CUTANA™ CUT&RUN Antibody/欣博盛生物

宿主来源Rabbit

实验应用CUT&RUN, ELISA, WB, IP, ICC

免疫原A synthetic peptide sequence (DYKDDDDK)

克隆性Monoclonal

保存温度自收到之日起,4℃下可稳定储存1年。

运输温度DO NOT FREEZE!! Frozen cold packs.


验证数据

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 1: Defined nucleosome spike-ins provide an in-assay control for DYKDDDDK Tag antibody in CUT&RUN

CUT&RUN was preformed as described in Figure 7. CUT&RUN sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the SNAP-CUTANA™ DYKDDDDK Tag Panel (EpiCypher 19-5001). Data are expressed as a percent relative to on-target recovery (DYKDDDDK Tag set to 100%) or total counts (IgG/GATA3). IgG/GATA3 show no preferential binding to unmodified or DYKDDDDK spike-in nucleosomes. DYKDDDDK Tag antibody selectively enriches the DYKDDDDK Tag spike-in nucleosome, validating the antibody in CUT&RUN.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 2: DYKDDDDK-tagged protein peaks in CUT&RUN

CUT&RUN was preformed as described in Figure 7. Heatmaps show DYKDDDDK Tag antibody-enriched peaks called for FLAG-GATA3 overexpressing cells (OE) in aligned rows relative to all other experimental conditions. Rows are ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal. A high degree of overlap is observed for DYKDDDDK and GATA3 antibodies as expected in the OE cells, while empty vector control (Ctrl) shows absence of FLAG and lower GATA3 enrichment representing endogenous protein. IgG shows low background signal. H3K4me3, a canonical mark of promoters, does not appear in regions of high GATA3 enrichment.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 3: CUT&RUN representative browser tracks for DYKDDDDK-tagged protein

CUT&RUN was preformed as described in Figure 7. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Two of the top called peaks in FLAG-GATA3 overexpressing (OE) cells are shown. The peaks show the same distribution patterns as observed in the genome-wide heatmaps (Figure 2). IgG shows low background, H3K4me3 is unchanged between empty vector control (Ctrl) and OE cells, GATA3 peaks are more robust in OE cells, and peaks overlap between GATA3 and DYKDDDDK antibodies in OE cells. These results demonstrate the robustness and specificity of DYKDDDDK Tag antibody in CUT&RUN experiments targeting FLAG-tagged proteins.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 4: Western blot data

Western analysis of FLAG-tagged protein in lysates from non-transfected human HEK293 cells, HEK293 transfected with Met-FLAG-IDO1, HEK293 transfected with IDO1-FLAG-HIS (Internal Tag), HEK293 transfected with IDO1-HIS-FLAG (C-Terminal Tag), and SUP-M2 cells. EpiCypher DYKDDDDK Tag antibody was used at 1:1,000. An HRP-conjugated goat anti-rabbit IgG antibody (Fortis A120-101P) was used as a secondary antibody. Chemiluminescence exposure time was 10 seconds. Lower panel shows rabbit anti-RPS3A antibody results (Fortis A305-003A).

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 5: Immunoprecipitation data

EpiCypher DYKDDDDK Tag antibody (20 µL/mg lysate) was used to immunoprecipitate lysates isolated from HEK293 cells transfected with either Met-FLAG-IDO1 (A) or IDO1-HIS-FLAG (C-terminal tag; B). For blotting immunoprecipitated DYKDDDDK Tag, EpiCypher DYKDDDDK Tag antibody was used at 1:1,000. Chemiluminescence exposure time was 10 seconds.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 6: Immunocytochemistry data

FFPE sections of HEK293 cells expressing either FLAG-tagged nuclear protein (left) or FLAG-tagged cytoplasmic protein (right) using EpiCypher DYKDDDDK Tag antibody. An HRP-conjugated goat anti-rabbit IgG antibody (Fortis A120-501P) was used as a secondary antibody. The substrate used was DAB.

EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

Figure 7: CUT&RUN methods

CUT&RUN was performed on 500k MDA-MB-231 native cells either stably overexpressing 3xFLAG-tagged GATA3 [1] or containing vector control. Either DYKDDDDK Tag (0.05 µg), H3K4me3 (0.5 µg; EpiCypher 13-0041), GATA3 (0.5 µg; CST 5852), or IgG (0.5 µg, EpiCypher 13-0042) antibodies were used with the CUTANA™ ChIC/CUT&RUN Kit v3 (EpiCypher 14-1048). Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth (vector control cells/overexpressing cells) was 5.6/4.5 million reads (IgG), 7.8/6.7 million reads (H3K4me3), 5.8/4.7 million reads (GATA3), and 6.2/5.5 million reads (DYKDDDDK Tag). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.


订购详情

货号

产品名称

规格

13-2031

DYKDDDDK Tag CUTANA™ CUT&RUN Antibody/欣博盛生物

100 µL


参考文献

[1] Takaku et al. Genome Biol. (2016). PMID: 26922637

 


EpiCypher新品推荐——DYKDDDDK Tag CUTANA™ CUT&RUN Antibody

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