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.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 5
#72364

SS18-SSX (E9X9V) XP® Rabbit mAb

Western Blotting Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using SS18-SSX (E9X9V) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). ASKA is a synovial sarcoma cell line with an SS18-SSX1 fusion, while SYO-1 is a synovial sarcoma cell line with an SS18-SSX2 fusion. As shown, SS18-SSX (E9X9V) XP® Rabbit mAb only detects the fusion protein and not WT proteins. Cell lysates were isolated and provided by the Kadoch laboratory.
Immunoprecipitation Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Immunoprecipitation of SS18-SSX (E9X9V) XP® Rabbit mAb from ASKA cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SS18-SSX (E9X9V) XP® Rabbit mAb. Western blot analysis was performed using SS18-SSX (E9X9V) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody. Cell lysates were isolated and provided by the Kadoch laboratory.
Immunohistochemistry Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human synovial sarcoma using SS18-SSX (E9X9V) XP® Rabbit mAb performed on the Leica® BOND Rx.
Immunohistochemistry Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded ASKA cell pellet (left, positive) or HeLa cell pellet (right, negative) using SS18-SSX (E9X9V) XP® Rabbit mAb. Cell pellets were isolated and provided by the Kadoch laboratory.
Immunohistochemistry Image 2: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma (left) and adenoid cystic carcinoma of the trachea (right) using SS18-SSX (E9X9V) XP® Rabbit mAb (upper) or SS18 (D6I4Z) Rabbit mAb #21792 (lower). Note the absence of the fusion protein in each carcinoma.
Immunohistochemistry Image 3: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human synovial sarcoma using SS18-SSX (E9X9V) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemistry Image 4: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human synovial sarcoma (left) and normal testis (right) using SS18-SSX (E9X9V) XP® Rabbit mAb (upper) or SSX (E5A2C) Rabbit mAb (Carboxy-terminal Antigen) #23855 (lower). Note that the fusion protein is not present in normal testis.
Chromatin Immunoprecipitation Image 1: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Aska cells and SS18-SSX (E9X9V) XP® Rabbit mAb, using SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina #56795. The figure shows binding across HES5, a known target gene of SS18-SSX1 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from Aska cells and SS18-SSX (E9X9V) XP® Rabbit mAb, using SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina #56795. The figure shows binding across chromosome 1 (upper), including HES5 (lower), a known target gene of SS18-SSX1 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: SS18-SSX (E9X9V) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from ASKA cells and either SS18-SSX (E9X9V) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HES5 Upstream Primers #59885, human SOX8 exon 3 primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one. Chromatin pellets were isolated and provided by the Kadoch laboratory.