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Datasheet
Monoclonal Antibody to cleaved ROCK-1
Cat.No IMG-383A
Description ROCK-1-cleaved
Format Purified
Unit 0.1 mg
Price(USD) 257.00
MSDS
Catalog No
:
IMG-383A
Contents
:
100 µg in 200 µl PBS containing 0.2% gelatin and 0.05% sodium azide. Sodium azide is highly toxic.
Isotype
:
MouseIgG
Clone
:
154C1465
Purification
:
Protein G Chromatography
Species
:
Chimpanzee, Dog, Human, Mouse, Rabbit, Rat
Host
:
Mouse
Application
Western blot analysis: 1-2 ug/ml Storage This antibody is supplied as protein-G purified protein. Store at 4°C, stable for 6 months. For long-term storage, store at -20°C.
Background The Rho target protein, Rho-associated coiled coil-containing protein kinase (ROCK)-1 has been found to be a new Caspase-3 substrate. ROCK, one of the effectors of the small GTPase Rho, has recently been shown to contribute significantly to myosin light chain (MLC) activation through two pathways: direct phosphorylation of MLC and phosphorylation of MLC phosphatase, leading to its inhibition. The increase in cellular contractility that is necessary for apoptotic membrane blebbing implies sustained augmentation of MLC phosphorylation. ROCK-1 consists of an amino-terminal kinase domain and an inhibitory cysteine/histidine-rich C-terminal domain that is located within a pleckstrin-homology region. They are joined by a variable region that contains the Rho-binding domain. During apoptosis, ROCK-I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. The caspase-3-mediated cleavage and activation of ROCK I induces phosphorylation of MLC and membrane blebbing, one of the first events in the execution phase of apoptosis.
Antigen
This antibody was generated by immunizing mice with a synthetic peptide sequence corresponding to amino acids 1114-1126 GNLPESRIEGWLS of human ROCK-1. This sequence is 100% conserved between human, chimpanzee, mouse, rat, dog, and rabbit. This immunogen peptide sequence is adjacent to the caspase-3 cleavage site of ROCK-1.
Application Notes
This mAb recognizes the cleaved C-terminus of ROCK-1. It does not recognize the full-length protein. Since ROCK-1 is cleaved by caspase-3 during apoptosis, this antibody may be used for detection of apoptosis.
Western blot analysis of cleaved ROCK-1 in A) untreated, B) 1 hour and C) 4 hour staurosporin-treated (2 uM) Jurkat cells using IMG-383A at 2 ug/ml. IMGENEX’s goat anti-mouse Ig HRP secondary antibody (IMGENEX, 20101) and PicoTect ECL substrate solution (IMGENEX, 10087K) were used for this test.
Reference 1. Amano, M. et al. J. Biol. Chem. 271, 20246-20249 (1996). 2. Sebbagh M, et al. Nat Cell Biol. 3(4):346-52 (2001). 3. Nakagawa O., et al. FEBS Lett. 392 (2), 189-193 (1996).
Product Citations 1. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells. Orlando KA, NL Stone, and RN Pittman. Experimental Cell Research 312:5-15 (2006).
Imgenex products cited:
1. ROCK-1 (Cleaved), IMG-383A: WB, Fig 3 (tamoxifen-treated Cos7 cells). IMG-383 detected a 30 kDa cleaved Rock-1 band in tamoxifen-treated treated cells. The addition of the Broad-Spectrum Caspase Inhibitor Z-VAD-FMK (IMI-2310-1, -5) prior to tamoxifen treatment blocked apoptosis induction and Rock-1 cleavage.
2. Z-VAD-FMK Pan Caspase Inhibitor, IMI-2310-1, -5): Inhibition of apoptosis in Cos7 cells, Figs 3, 6A.
2. Rho kinase regulates phagocytosis, surface expression of GlcNAc, and Golgi fragmentation of apoptotic PC12 cells. Orlando KA and RN Pittman. Experimental Cell Research 312: 3298-3311 (2006) .
Imgenex products cited:
1. ROCK-1 (Cleaved), IMG-383A: WB, Figs 1,2 (serum-starved PC12 cells). IMG-383 detected a 30 kDa cleaved Rock-1 band in serum-starved PC12 cells. The addition of the Broad-Spectrum Caspase Inhibitor Z-VAD-FMK (IMI-2310-1, -5) during serum starvation did not block Rock-1 cleavage.
2. Z-VAD-FMK Pan Caspase Inhibitor, IMI-2310-1, -5): Figs 1,2: Inhibition of caspase activation in PC12 cells, Figs 1, 2. 3. Anoxia and reoxygenation of human endothelial cells decreases ceramide glucosyltransferase expression and activates caspases. Zhao H, M Miller, K Pfeiffer, JA Buras and GL Stahl. FASAB J 10.1096/fj02-806fje. (2003). WB (Normoxic/anoxic/reoxygenated HUVEC cells), Fig 1.
Research purposes only.
Not for diagnostic or in vivo use. This product is guaranteed to perform as indicated on the datasheet for one year from the date of purchase.
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