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EpiCypher新品推薦——DYKDDDDK Tag CUTANA? CUT&RUN Antibody

2024-03-01
瀏覽次數(shù): 17

EpiCypher新推出的DYKDDDDK抗體,適用于利用FLAG?*標(biāo)記的靶蛋白進行的研究。該抗體符合EpiCypher的“CUTANA Compatible”標(biāo)準(zhǔn),可用于研究蛋白質(zhì)-DNA互作的新技術(shù),CUT & RUN和CUT & Tag。每個批次的CUTANA Compatible抗體均使用經(jīng)EpiCypher優(yōu)化的方案(https://www.epicypher.com/resources/protocols/),按照指定的方法進行測試,并確定產(chǎn)生的峰值所顯示的基因組分布模式與已報道的靶標(biāo)功能一致。在過表達 3xFLAG-GATA3的乳腺癌細胞中,該DYKDDDDK Tag 抗體產(chǎn)生與 GATA3 抗體高度重疊的 CUT&RUN 峰(圖 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.



產(chǎn)品詳情

產(chǎn)品名稱:DYKDDDDK Tag CUTANA? CUT&RUN Antibody

宿主來源:Rabbit

實驗應(yīng)用:CUT&RUN, ELISA, WB, IP, ICC

免疫原:A synthetic peptide sequence (DYKDDDDK)

克隆性:Recombinant?Monoclonal?

保存條件:Stable for 1 year at 4°C from date of receipt


驗證數(shù)據(jù)

EpiCypher新品推薦——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新品推薦——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新品推薦——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新品推薦——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新品推薦——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新品推薦——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新品推薦——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.


訂購詳情

貨號

產(chǎn)品名稱

規(guī)格

13-2031

DYKDDDDK Tag CUTANA? CUT&RUN Antibody

100 μL


參考文獻

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

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EpiCypher新品推薦——DYKDDDDK Tag CUTANA? CUT&RUN Antibody

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