Revision 8
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

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

62

Source/Isotype:

Rabbit IgG

UniProt ID:

#O96017

Entrez-Gene Id:

11200

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:50 - 1:250
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:100 - 1:400
Flow Cytometry (Fixed/Permeabilized) 1:50 - 1:200

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 #28130.

Specificity / Sensitivity

Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb detects endogenous levels of Chk2 only when phosphorylated at Thr68. Non-specific staining of mitotic cells was observed by immunohistochemistry.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr68 of human Chk2.

Background

Chk2 is the mammalian orthologue of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases (1-3). The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues (Ser19, Thr26, Ser28, Ser33, Ser35, Ser50, and Thr68) each followed by glutamine (SQ or TQ motif). These are known to be preferred sites for phosphorylation by ATM/ATR kinases (4,5). After DNA damage by ionizing radiation (IR), UV irradiation, or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (5-7). The SQ/TQ cluster domain, therefore, seems to have a regulatory function. Phosphorylation at Thr68 is a prerequisite for the subsequent activation step, which is attributable to autophosphorylation of Chk2 at residues Thr383 and Thr387 in the activation loop of the kinase domain (8).

  1. Allen, J.B. et al. (1994) Genes Dev. 8, 2401-2415.
  2. Weinert, T.A. et al. (1994) Genes Dev. 8, 652-665.
  3. Murakami, H. and Okayama, H. (1995) Nature 374, 817-819.
  4. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
  5. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
  6. Melchionna, R. et al. (2000) Nat. Cell Biol. 2, 762-765.
  7. Ahn, J.Y. et al. (2000) Cancer Res. 60, 5934-5936.
  8. Lee, C.H. and Chung, J.H. (2001) J. Biol. Chem. 276, 30537-30541.

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)

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.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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 8
#2197

Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb

Western Blotting Image 1: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa cells, untreated or UV-treated, using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
Western Blotting Image 1: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from HEK 293 cells treated with UV (50 mJ, 30 min recovery) using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb #2197. The virtual lane view (left) shows a single 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: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Immunoprecipitation of phospho-chk2 from UV-treated HT29 cells using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb followed by western blot using the same antibody.
Immunohistochemistry Image 1: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
Immunohistochemistry Image 2: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
Immunohistochemistry Image 3: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
Immunohistochemistry Image 4: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HCT 116 cell pellets, control (top) or CRISPR/Cas9 Chk2 knockout (KO) (bottom), untreated (left) or UV-treated (100 mJ, 2 hr recovery, right) using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.
Flow Cytometry Image 1: Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb Expand Image
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100mJ/cm2, 2 hr recovery; green) using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) was used as a secondary antibody.