Revision 5

#23064Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) orders@cellsignal.com

Support: 877-678-TECH (8324)

Web: info@cellsignal.com cellsignal.com

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

WB, IP, IF-F, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

35

Source/Isotype:

Rabbit IgG

UniProt ID:

#P48431

Entrez-Gene Id:

6657

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.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Frozen) 1:400
Immunofluorescence (Immunocytochemistry) 1:400
Flow Cytometry (Fixed/Permeabilized) 1:200 - 1:800
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 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

Sox2 (D9B8N) Rabbit mAb recognizes endogenous levels of total Sox2 protein. The abundant nonspecific cytoplasmic labeling was observed in adult brain by immunofluorescence (IF). However, the specific staining was observed in embryonic tissue, including brain, by IF.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala188 of human Sox2 protein.

Background

Embryonic stem cells (ESC) derived from the inner cell mass of the blastocyst are unique in their pluripotent capacity and potential for self-renewal (1). Research studies demonstrate that a set of transcription factors that includes Oct-4, Sox2, and Nanog forms a transcriptional network that maintains cells in a pluripotent state (2,3). Chromatin immunoprecipitation experiments show that Sox2 and Oct-4 bind to thousands of gene regulatory sites, many of which regulate cell pluripotency and early embryonic development (4,5). siRNA knockdown of either Sox2 or Oct-4 results in loss of pluripotency (6). Induced overexpression of Oct-4 and Sox2, along with additional transcription factors Klf4 and c-Myc, can reprogram both mouse and human somatic cells to a pluripotent state (7,8). Additional evidence demonstrates that Sox2 is also present in adult multipotent progenitors that give rise to some adult epithelial tissues, including several glands, the glandular stomach, testes, and cervix. Sox2 is thought to regulate target gene expression important for survival and regeneration of these tissues (9).

  1. Conley, B.J. et al. (2004) Int J Biochem Cell Biol 36, 555-67.
  2. Pesce, M. and Schöler, H.R. (2001) Stem Cells 19, 271-8.
  3. Pan, G. and Thomson, J.A. (2007) Cell Res 17, 42-9.
  4. Boyer, L.A. et al. (2005) Cell 122, 947-56.
  5. Loh, Y.H. et al. (2006) Nat Genet 38, 431-40.
  6. Matin, M.M. et al. (2004) Stem Cells 22, 659-68.
  7. Takahashi, K. and Yamanaka, S. (2006) Cell 126, 663-76.
  8. Okita, K. et al. (2007) Nature 448, 313-7.
  9. Arnold, K. et al. (2011) Cell Stem Cell 9, 317-29.

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 IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) 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.

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
#23064

Sox2 (D9B8N) Rabbit mAb

Western Blotting Image 1: Sox2 (D9B8N) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines, using Sox2 (D9B8N) Rabbit mAb.
Immunoprecipitation Image 1: Sox2 (D9B8N) Rabbit mAb Expand Image
Immunoprecipitation of Sox2 from F9 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Sox2 (D9B8N) Rabbit mAb. Western blot analysis was performed using Sox2 (D9B8N) Rabbit mAb.
Immunofluorescence Image 1: Sox2 (D9B8N) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of developing mouse E12 foregut (left), spinal cord and somites (middle), or brain (right) using Sox2 (D9B8N) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunofluorescence Image 1: Sox2 (D9B8N) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of F9 (left), 3T3 (middle left), NCCIT (middle right), or HeLa (right) cells using Sox2 (D9B8N) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
Flow Cytometry Image 1: Sox2 (D9B8N) Rabbit mAb Expand Image
Flow cytometric analysis of HeLa cells (blue) and NTERA cells (green) using Sox2 (D9B8N) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 2: Sox2 (D9B8N) Rabbit mAb Expand Image
Flow cytometric analysis of NIH3T3 cells (blue) and F9 cells (green) using Sox2 (D9B8N) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: Sox2 (D9B8N) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES cells and Sox2 (D9B8N) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across POU5F1/OCT4, a known target gene of Sox2 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: Sox2 (D9B8N) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES cells and Sox2 (D9B8N) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 17 (upper), including POU5F1/OCT4 (lower), a known target gene of Sox2 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: Sox2 (D9B8N) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES cells and either Sox2 (D9B8N) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse Oct-4 Promoter Primers #4653, SimpleChIP® Mouse XIST Intron 1 Primers #4659, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. 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.
CUT and RUN Image 1: Sox2 (D9B8N) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and Sox2 (D9B8N) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across Nanog, a known target gene of Sox2 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: Sox2 (D9B8N) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and Sox2 (D9B8N) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 12 (upper), including Nanog (lower), a known target gene of Sox2 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: Sox2 (D9B8N) Rabbit mAb Expand Image
CUT&RUN was performed with NCCIT cells and either Sox2 (D9B8N) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human Nanog Promoter Primers #95064, SimpleChIP® Human Oct-4 Promoter Primers #4641, 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.