Revision 9

#42427Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, FC-FP, ChIP, ChIP-seq, C&R, C&T

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

58, 63-70, 85

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9UKS7

Entrez-Gene Id:

22807

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.

The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:200
Flow Cytometry (Fixed/Permeabilized) 1:50 - 1:200
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50
CUT&Tag 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Helios (D8W4X) XP® Rabbit mAb recognizes endogenous levels of total Helios protein.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly73 of human Helios protein.

Background

Helios (Ikaros family zinc finger 2, IKZF2) is an Ikaros family transcription factor composed of several zinc fingers that mediate DNA binding and homodimerization or heterodimerization with other Ikaros family proteins (1,2). In the hematopoietic system, Helios expression is restricted to T cells and early hematopoietic progenitors (1,2). In regulatory T cells, expression of Helios contributes to an anergic phenotype by binding to the IL-2 promoter and suppressing IL-2 transcription (3). In addition, alteration of the corresponding Helios gene IKZF2 is one hallmark of low-hypodiploid acute lymphoblastic leukemia (4).

  1. Hahm, K. et al. (1998) Genes Dev 12, 782-96.
  2. Kelley, C.M. et al. (1998) Curr Biol 8, 508-15.
  3. Baine, I. et al. (2013) J Immunol 190, 1008-16.
  4. Holmfeldt, L. et al. (2013) Nat Genet 45, 242-52.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 9
#42427

Helios (D8W4X) XP® Rabbit mAb

Western Blotting Image 1: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 Helios (D8W4X) XP® Rabbit mAb(上)或 β-Actin (D6A8) Rabbit mAb #8457(下)对不同细胞系的提取物进行蛋白质印迹分析。
Western Blotting Image 2: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 Helios (D8W4X) XP® Rabbit mAb 对未转染 (-),或构建表达 Myc 标记的全长人 Helios (hHelios-Myc; +) 转染的 293T 细胞提取物进行蛋白质印迹分析。
Western Blotting Image 1: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 Helios (D8W4X) XP® Rabbit mAb #42427 对 Jurkat 细胞的裂解物 (0.1 mg/mL) 进行 Simple Western™ 分析。虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。
Immunoprecipitation Image 1: Helios (D8W4X) XP® Rabbit mAb Expand Image
对来自 Jurkat 细胞提取物的 Helios 进行免疫沉淀法分析。泳道 1 为10% 输入,泳道2为 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900,而泳道3为 Helios (D8W4X) XP® Rabbit mAb。使用 Helios (D8W4X) XP® Rabbit mAb 执行蛋白质印迹分析。
Flow Cytometry Image 1: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 Helios (D8W4X) XP® Rabbit mAb 对 RL(蓝色)和 Jurkat(绿色) 细胞进行流式细胞术分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 用作二抗。
Chromatin Immunoprecipitation Image 1: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 SimpleChIP® 酶解染色质免疫共沉淀试剂盒(磁珠法) #9005 对 Jurkat 细胞的交联染色质和 Helios (D8W4X) XP® 兔单克隆抗体进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 Helios 的已知靶基因 IKZF3 内结合(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 2: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 SimpleChIP® 酶解染色质免疫共沉淀试剂盒(磁珠法) #9005 对 Jurkat 细胞的交联染色质和 Helios (D8W4X) XP® 兔单克隆抗体进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体 17(上图)内的结合,包括 Helios 的已知靶标基因 IKZF3(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: Helios (D8W4X) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Jurkat cells and either Helios (D8W4X) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. 使用 SimpleChIP® Human IKZF3 Promoter Primers #99663、human IL-2 promoter primers 和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 定量。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
CUT and RUN Image 1: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 Jurkat 细胞和 Helios (D8W4X) XP® Rabbit mAb 进行 CUT&RUN 检测。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示在 IKZF3 基因内结合。
CUT and RUN Image 2: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 Jurkat 细胞和 Helios (D8W4X) XP® Rabbit mAb 进行 CUT&RUN 检测。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示在染色体 17(上图)内的结合,包括 IKZF3 基因(下图)。
CUT and RUN Image 3: Helios (D8W4X) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 Jurkat 细胞和 Helios (D8W4X) XP® Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN 分析。使用 human TMEM exon 1、human CKS1B exon 1 和 SimpleChIP® Human α Satellite Repeat Primers #4486 进行实时 PCR 来对富集的 DNA 进行定量分析。每个样本中免疫沉淀 DNA 的数量表示为相对于输入染色质总量的信号,相当于
CUT and Tag Image 1: Helios (D8W4X) XP® Rabbit mAb Expand Image
CUT&Tag was performed with Jurkat cells and Helios (D8W4X) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across IKZF3, a known target gene of Helios (see our ChIP-qPCR figure).
CUT and Tag Image 2: Helios (D8W4X) XP® Rabbit mAb Expand Image
CUT&Tag was performed with Jurkat cells and Helios (D8W4X) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 17 (upper), including IKZF3 (lower), a known target gene of Helios (see our ChIP-qPCR figure).