Revision 8

#15103Store 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, IHC-P, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

50,58,65

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9UKT9

Entrez-Gene Id:

22806

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 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.
Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:200
Immunohistochemistry (Paraffin) 1:50
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:100
Chromatin IP-seq 1:100
CUT&RUN 1:100

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.

For a carrier free (BSA and azide free) version of this product see product #97279.

Specificity / Sensitivity

Aiolos (D1C1E) Rabbit mAb recognizes endogenous levels of total Aiolos protein.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro46 of human Aiolos protein.

Background

Aiolos is an Ikaros family transcription factor composed of several zinc fingers that mediate DNA binding and homodimerization or heterodimerization with other Ikaros family members (1). Multiple Aiolos isoforms are generated through alternative splicing of the portion of the transcript encoding the amino-terminal zinc fingers (2). Aiolos is expressed by lymphoid tissues, with highest expression levels seen in mature B and T cells (1). Ikaros family proteins control lymphocyte development by recruiting chromatin remodeling complexes to DNA (3). B cells from mice lacking Aiolos have a reduced threshold for activation, increased proliferation, and elevated levels of IgG and IgE. In addition, Aiolos mice develop B cell lymphomas (4). In T cells, Aiolos contributes to Th17 cell differentiation by suppressing IL-2 expression (5). Aberrant expression of Aiolos in transformed epithelial cells promotes anchorage independence through downregulation of adhesion-related genes (6). Alterations in the Aiolos gene are observed in near haploid acute lymphoblastic leukemia, and the genetic locus containing Aiolos is linked to increased susceptibility to rheumatoid arthritis and systemic lupus erythematosus (7-9).

  1. Morgan, B. et al. (1997) EMBO J 16, 2004-13.
  2. Liippo, J. et al. (2001) Eur J Immunol 31, 3469-74.
  3. Kim, J. et al. (1999) Immunity 10, 345-55.
  4. Wang, J.H. et al. (1998) Immunity 9, 543-53.
  5. Quintana, F.J. et al. (2012) Nat Immunol 13, 770-7.
  6. Li, X. et al. (2014) Cancer Cell 25, 575-89.
  7. Holmfeldt, L. et al. (2013) Nat Genet 45, 242-52.
  8. Kurreeman, F.A. et al. (2012) Am J Hum Genet 90, 524-32.
  9. Lessard, C.J. et al. (2012) Am J Hum Genet 90, 648-60.

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 IHC-P: Immunohistochemistry (Paraffin) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

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 8
#15103

Aiolos (D1C1E) Rabbit mAb

Western Blotting Image 1: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 Aiolos (D1C1E) Rabbit mAb(上)和 β-Actin (D6A8) Rabbit mAb #8547(下)对未处理 (-) 或经来那度胺处理(2 μM,24 小时;+),对 U-937 细胞和 RPMI-8226 细胞提取物进行蛋白质印迹分析。
Western Blotting Image 2: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 Aiolos (D1C1E) Rabbit mAb 对不同细胞系提取物进行蛋白质印迹分析。
Western Blotting Image 1: Aiolos (D1C1E) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (1.0 mg/mL) from RPMI-8226 cells using Aiolos (D1C1E) Rabbit mAb #15103. 虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标条带(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: Aiolos (D1C1E) Rabbit mAb Expand Image
对 HT-29 细胞提取物 Aiolos 进行免疫沉淀。泳道 1 为 10% input,泳道 2 为 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900,泳道 3 为 Aiolos (D1C1E) Rabbit mAb。使用 Aiolos (D1C1E) Rabbit mAb 进行蛋白质印迹分析。
Immunohistochemistry Image 1: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 Aiolos (D1C1E) Rabbit mAb 对石蜡包埋的人非小细胞肺癌进行免疫组织化学分析。
Immunohistochemistry Image 2: Aiolos (D1C1E) Rabbit mAb Expand Image
在对照肽(左)或抗原特异性肽(右)存在的情况下,使用 Aiolos (D1C1E) Rabbit mAb 对石蜡包埋的人卵巢癌进行免疫组织化学分析。
Flow Cytometry Image 1: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 Aiolos (D1C1E) Rabbit mAb,对 U-937 细胞(蓝色)、未处理的 RPMI-8226 细胞(绿色)或经来那度胺处理的 RPMI-8226 细胞(2 μM,24 小时;红色)进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。
Flow Cytometry Image 2: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 Aiolos (D1C1E) Rabbit mAb 对经固定(2% 甲醛)和通透(0.1% Triton X-100)处理且用 CD19 antibody 共染色的小鼠脾细胞进行流式细胞分析。Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 用作二抗。
Chromatin Immunoprecipitation Image 1: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对Raji 细胞中提取的交联染色质在加入 Aiolos (D1C1E) Rabbit mAb 后,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 SLC22A7 基因内结合。
Chromatin Immunoprecipitation Image 2: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对Raji 细胞中提取的交联染色质在加入 Aiolos (D1C1E) Rabbit mAb 后,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 NPR3(上图)内的结合,包括 SLC22A7 基因(下图)。
Chromatin Immunoprecipitation Image 3: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 对Raji 细胞中提取的交联染色质,在加入Aiolos (D1C1E) Rabbit mAb 或 Normal Rabbit IgG #2729后,进行染色质免疫沉淀。使用 SimpleChIP® Human HSP90β Promoter Primers #12899、human SirT1 promoter primers 和 SimpleChIP® Human α Satellite Repeat Primers #4486 并通过实时 PCR 对富集的 DNA 进行定量分析。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: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 Raji 细胞和 Aiolos (D1C1E) 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 Aiolos 的已知靶标基因 ARID1A 基因内的结合(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 2: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 Raji 细胞和 Aiolos (D1C1E) 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 1(上图)内的结合,包括 Aiolos 的已知靶标基因 ARID1A 基因(下图)(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 3: Aiolos (D1C1E) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对未经处理的€‚ Raji 细胞和 Aiolos (D1C1E) Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN 检测。使用人 ARID1A 启动子引物、人 SYF2 上游引物和人 KC6 上游引物通过实时 PCR 对富集的 DNA 进行定量。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。