Revision 3

#28999Store 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, IHC-P, IF-IC

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

160

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q13464

Entrez-Gene Id:

6093

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Immunofluorescence (Immunocytochemistry) 1:400 - 1:1600

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

ROCK1 (E2G4N) Rabbit mAb recognizes endogenous levels of total ROCK1 protein. Non-specific staining was observed in placenta by immunohistochemistry. In some cell lines, this antibody cross-reacts with 45-55 kDa bands of unknown origin by western blot.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the central region of human ROCK1 protein.

Background

ROCK (Rho-associated kinase), a family of serine/threonine kinases, is an important downstream target of Rho-GTPase and plays an important role in Rho-mediated signaling. Two isoforms of ROCK have been identified: ROCK1 and ROCK2. ROCK is composed of N-terminal catalytic, coiled-coil, and C-terminal PH (pleckstrin homology) domains. The C-terminus of ROCK negatively regulates its kinase activity (1,2). ROCK1 is cleaved by caspase-3 at a conserved DETD1113/G sequence resulting in loss of its C-terminal inhibitory domain (3). ROCK2 is directly cleaved by granzyme B (grB). Cleavage activates ROCK and leads to phosphorylation of myosin light chain (MLC) and inhibition of myosin phosphatase (4). This phosphorylation may account for the mechanism by which Rho regulates cytokinesis, cell motility, cell membrane blebbing during apoptosis, and smooth muscle contraction (5-7).

  1. Nakagawa, O. et al. (1996) FEBS Lett. 392, 189-193.
  2. Lee, J.H. et al. (2004) J. Cell. Biol. 167, 327-337.
  3. Sebbagh, M. et al. (2001) Nat Cell Biol 3, 346-52.
  4. Sebbagh, M. et al. (2005) J Exp Med 201, 465-71.
  5. Amano, M. et al. (1996) J Biol Chem 271, 20246-9.
  6. Kureishi, Y. et al. (1997) J Biol Chem 272, 12257-60.
  7. Totsukawa, G. et al. (2000) J Cell Biol 150, 797-806.

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 nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry)

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.
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 3
#28999

ROCK1 (E2G4N) Rabbit mAb

Western Blotting Image 1: ROCK1 (E2G4N) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using ROCK1 (E2G4N) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Low expression of ROCK1 protein in KATO III cells is consistent with the predicted expression pattern.
Western Blotting Image 1: ROCK1 (E2G4N) Rabbit mAb Expand Image

Simple Western™ analysis of lysates (0.1 mg/mL) from C6 cells using ROCK1 (E2G4N) Rabbit mAb #28999. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (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.

Immunohistochemistry Image 1: ROCK1 (E2G4N) Rabbit mAb Expand Image

Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using ROCK1 (E2G4N) Rabbit mAb.

Immunohistochemistry Image 2: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 3: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human urothelial carcinoma using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 4: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human B-cell non-Hodgkin lymphoma using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 5: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human papillary thyroid carcinoma using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 6: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 7: ROCK1 (E2G4N) Rabbit mAb Expand Image

Immunohistochemical analysis of paraffin-embedded normal human esophagus using ROCK1 (E2G4N) Rabbit mAb.

Immunohistochemistry Image 8: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human small intestine using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 9: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded 4T1 syngeneic tumor using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 10: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal mouse testis using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 11: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal mouse lung using ROCK1 (E2G4N) Rabbit mAb.
Immunohistochemistry Image 12: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human kidney using ROCK1 (E2G4N) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemistry Image 13: ROCK1 (E2G4N) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded SU-DHL-4 cell pellet (left, positive) or KATO III cell pellet (right, negative) using ROCK1 (E2G4N) Rabbit mAb.
Immunofluorescence Image 1: ROCK1 (E2G4N) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of MCF7 cells (left, positive) and KATO III cells (right, negative) using ROCK1 (E2G4N) Rabbit mAb (green) and DAPI #4083 (blue).