Revision 5

#72364Store 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, IP, IHC-Bond, IHC-P, ChIP, ChIP-seq

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

65, 75

Source/Isotype:

Rabbit IgG

UniProt ID:

#A4PIV8

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 × 10^6 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
IHC Leica Bond 1:200 - 1:800
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Chromatin IP 1:50
Chromatin IP-seq 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.

For a carrier-free (BSA and azide free) version of this product see product #92761.

Specificity / Sensitivity

SS18-SSX (E9X9V) XP® Rabbit mAb recognizes endogenous levels of total SS18-SSX fusion protein. This antibody does not cross-react with wild-type SS18 or SSX proteins.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the fusion site of human SS18-SSX protein.

Background

SS18 is a protein that has been shown to be a part of the SWI/SNF complex (1, 2). The SS18-SSX fusion proteins are a result of in-frame fusions that fuse the SS18 gene on chromosome 18 with X chromosome genes SSX1, SSX2, and to a lesser extent SSX4 (3). Human synovial sarcoma (SS) accounts for 8-10% of all soft tissue malignancies and 95% of these malignancies express the recurrent translocation of the SS18 gene on chromosome 18 (3). The N-terminal SNH domain (SYT N-terminal homology domain) of the SS18 protein interacts with SWI/SNF chromatin remodeling complexes via the N terminal region of BRM and BRG1 subunits (4). Studies of the SS18-SSX fusion in SS suggest that endogenous SS18 competes with the mutant SS18-SSX fusion for occupancy in the SWI/SNF complexes resulting in the displacement of SNF5 (BAF47) subunit. Displacement of the SNF5 subunit results in altered function of the SWI/SNF complex that leads to deregulated expression of genes such as Sox2 in SS (3).

  1. Nagai, M. et al. (2001) Proc Natl Acad Sci U S A 98, 3843-8.
  2. Thaete, C. et al. (1999) Hum Mol Genet 8, 585-91.
  3. Kadoch, C. and Crabtree, G.R. (2013) Cell 153, 71-85.
  4. Perani, M. et al. (2003) Oncogene 22, 8156-67.

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 IP: Immunoprecipitation IHC-Bond: IHC Leica Bond IHC-P: Immunohistochemistry (Paraffin) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq

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.
SignalStain is a registered trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
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 5
#72364

SS18-SSX (E9X9V) XP® Rabbit mAb

Western Blotting Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用 SS18-SSX (E9X9V) XP® Rabbit mAb(上图)或 β-Actin (D6A8) Rabbit mAb #8457(下图),对不同细胞系提取物进行蛋白质印迹分析。ASKA 是具有 SS18-SSX1 融合的滑膜肉瘤细胞系,而 SYO-1 是具有 SS18-SSX2 融合的滑膜肉瘤细胞系。如图所示,SS18-SSX (E9X9V) XP® Rabbit mAb 仅检测融合蛋白,而不检测 WT 蛋白。由 Kadoch 实验室提供并分离细胞裂解物。
Immunoprecipitation Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用 SS18-SSX (E9X9V) XP® Rabbit mAb 对 ASKA 细胞提取物进行免疫沉淀分析。泳道 1 为 10% input,泳道 2 为 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900,泳道 3 为 SS18-SSX (E9X9V) XP® Rabbit mAb。使用 SS18-SSX (E9X9V) XP® Rabbit mAb 进行蛋白质印迹分析。Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 用作二抗。由 Kadoch 实验室提供并分离细胞裂解物。
Immunohistochemistry Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
在 Leica® BOND Rx 上使用 SS18-SSX (E9X9V) XP® Rabbit mAb 对石蜡包埋的人滑膜肉瘤进行免疫组织化学分析。
Immunohistochemistry Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用 SS18-SSX (E9X9V) XP® Rabbit mAb 对石蜡包埋的 ASKA 细胞沉淀物(左图,阳性)或 HeLa 细胞沉淀物(右图,阴性)进行免疫组织化学分析。由 Kadoch 实验室提供并分离细胞沉淀物。
Immunohistochemistry Image 2: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用 SS18-SSX (E9X9V) XP® Rabbit mAb(上图)或SS18 (D6I4Z) Rabbit mAb #21792 对石蜡包埋的人前列腺癌(左图)和气管腺样囊性癌(右图)进行免疫组织化学分析注意:每种癌中都没有融合蛋白。
Immunohistochemistry Image 3: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
以浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(右图)作为对照,使用 SS18-SSX (E9X9V) XP® Rabbit mAb(左图)对石蜡包埋的人滑膜肉瘤进行免疫组织化学分析。
Immunohistochemistry Image 4: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用 SS18-SSX (E9X9V) XP® Rabbit mAb(上图)或 SSX (E5A2C) Rabbit mAb (Carboxy-terminal Antigen) #23855(下图)对石蜡包埋的人滑膜肉瘤(左图)和正常睾丸(右图)进行免疫组织化学分析。请注意,正常睾丸细胞中无融合蛋白。
Chromatin Immunoprecipitation Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用 SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对 Aska 细胞中的交联染色质和 SS18-SSX (E9X9V) XP® Rabbit mAb 进行染色质免疫沉淀分析。使用 DNA Library Prep Kit for Illumina #56795 制备 DNA 库。图片中显示在 HES5 内的结合,而 HES5 是已知的 SS18-SSX1 靶标基因(请参阅含有 ChIP-qPCR 数据的附图)。
Chromatin Immunoprecipitation Image 2: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用 SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对 Aska 细胞中的交联染色质和 SS18-SSX (E9X9V) XP® Rabbit mAb 进行染色质免疫沉淀分析。使用 DNA Library Prep Kit for Illumina #56795 制备 DNA 库。结果图显示在染色体 1(上图)内的结合,包括 SS18-SSX1 的已知靶标基因 HES5(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
使用SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对 ASKA 细胞的交联染色质与 SS18-SSX (E9X9V) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 进行染色质免疫沉淀分析。使用 SimpleChIP® Human HES5 Upstream Primers #59885、human SOX8 exon 3 primers 和 SimpleChIP® Human α Satellite Repeat Primers #4486,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。由 Kadoch 实验室提供并分离染色质沉淀物。