Revision 5

#94205Store 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, ChIP

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

90

Source/Isotype:

Rabbit IgG

UniProt ID:

#P42229, #P51692

Entrez-Gene Id:

6776, 6777

Product Information

Product Usage Information

For optimal ChIP 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.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:50 - 1:250
Immunoprecipitation 1:200
Chromatin IP 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

Stat5 (D2O6Y) Rabbit mAb recognizes endogenous levels of total Stat5 protein. This antibody recognizes both alpha and beta isoforms of Stat5 (Stat5a and Stat5b).

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu60 of human Stat5 protein.

Background

Stat5 is activated in response to a wide variety of ligands, including IL-2, GM-CSF, growth hormone, and prolactin. Phosphorylation at Tyr694 is obligatory for Stat5 activation (1,2). This phosphorylation is mediated by Src upon erythropoietin stimulation (3). Stat5 is constitutively active in some leukemic cell types (4). Phosphorylated Stat5 is found in some endothelial cells treated with IL-3, which suggests its involvement in angiogenesis and cell motility (5). Stat5a and Stat5b are independently regulated and activated in various cell types. For instance, interferon treatment predominantly activates Stat5a in U-937 cells and Stat5b in HeLa cells (6).

  1. Gouilleux, F. et al. (1994) EMBO J 13, 4361-9.
  2. Wakao, H. et al. (1994) EMBO J 13, 2182-91.
  3. Okutani, Y. et al. (2001) Oncogene 20, 6643-50.
  4. Demoulin, J.B. et al. (1999) J Biol Chem 274, 25855-61.
  5. Dentelli, P. et al. (1999) J Immunol 163, 2151-9.
  6. Meinke, A. et al. (1996) Mol Cell Biol 16, 6937-44.

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 ChIP: Chromatin IP

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.
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
#94205

Stat5 (D2O6Y) Rabbit mAb

Western Blotting Image 1: Stat5 (D2O6Y) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using Stat5 (D2O6Y) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 2: Stat5 (D2O6Y) Rabbit mAb Expand Image
Western blot analysis of PC-3 cells, mock transfected (-) or transfected with constructs expressing full-length human Stat5a (+) or Stat5b (+) using Stat5 (D2O6Y) Rabbit mAb.
Western Blotting Image 1: Stat5 (D2O6Y) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from HDLM-2 cells using Stat5 (D2O6Y) Rabbit mAb #94205. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western Blotting Image 2: Stat5 (D2O6Y) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from HDLM-2 cells using Stat5 (D2O6Y) Rabbit mAb #94205. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: Stat5 (D2O6Y) Rabbit mAb Expand Image
Immunoprecipitation of Stat5 from K-562 cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Stat5 (D2O6Y) Rabbit mAb. Western blot was performed using Stat5 (D2O6Y) Rabbit mAb.
Chromatin Immunoprecipitation Image 1: Stat5 (D2O6Y) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from BaF3 cells starved of IL-3 for 6 hours followed by induction with IL-3 for 45 minutes, and either Stat5 (D2O6Y) 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 CIS Intron 1 Primers #5131, mouse SOCS-3 promoter primers, 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.