ROCK Activity Immunoblot Kit

Produktnummer ABIN2345099

Kurzübersicht für ROCK Activity Immunoblot Kit (ABIN2345099)

Applikation: Western Blotting (WB)

Produktdetails

Produktmerkmale: ROCK Activity Immunoblot Kit utilizes recombinant MYPT1 as ROCK substrate. After incubating the substrate with ROCK samples (such as purified kinase, cell lysate or immunoprecipitate), the phosphorylated MYPT1 is detected by western blot analysis using an anti- phospho-MYPT1 (Thr696) (Figure 1). ROCK Activity Immunoblot Kit provides a simple and fast tool to monitor ROCK activity using its physiological substrate. The kit also includes active ROCK-II as a positive control. Each kit provides sufficient quantities to perform 20 assays. Related Products 1. STA-416: 96-well ROCK Activity Assay Kit 2. STA-400: Ras Activation Assay Kit 3. STA-402: Cdc42 Activation Assay Kit 4. STA-403: Rho Activation Assay Kit 5. STA-404: Rac/Cdc42 Activation Assay Combo Kit 6. STA-405: Rho/Rac/Cdc42 Activation Assay Combo Kit 7. STA-410: PAK1 PBD Agarose Beads 8. STA-411: Raf1 PBD Agarose Beads 9. STA-412: Rhotekin PBD Agarose Beads 10. STA-452: GFP-RhoA Expression Vector Set 11. STA-456: RhoA Expression Vector Set 12. STA-460: Exoenzyme C3 (Rho Inhibitor) Expression Vector 2

Bestandteile:

1. ROCK Substrate : One 40 μL vial containing 0.25 mg/mL recombinant MYPT1
2. 10X Kinase Buffer : Three 1.0 mL vials of 250 mM Tris, pH 7.5, 100 mM MgCl2, 50 mM Glycerol-2-Phsophate, 1 mM Na3VO4
3. ATP Solution : One 400 μL vial of 10 mM ATP
4. Anti-phospho-MYPT1 (Thr696) : One 50 μL vial
5. Goat Anti-Rabbit IgG, HRP-conjugate : One 100 μL vial
6. Active ROCK-II : One 20 μL vial containing 10 ng active ROCK-II in 25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM Glycerol-2-Phosphate, 0.1 mM Na3VO4, 10% Glycerol, 0.1% BSA

Benötigtes Material:

1. ROCK sample (purified kinase, cell lysate or immunoprecipitate)
2. DTT
3. 30 °C incubator or water bath
4. 4X SDS-PAGE sample buffer
5. Electrophoresis and immunoblotting systems
6. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05 % Tween-20)
7. Immunoblotting blocking buffer (TBST containing 5 % Non-fat Dry Milk)
8. PVDF or nitrocellulose membrane
9. ECL Detection Reagents

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Safe non-radioactive Western blot assay format

Aufbereitung der Reagenzien:

• 1X Kinase Buffer: Dilute to 10X Kinase Buffer to 1X in deionized water. 1X Kinase Buffer may be stored at 4 °C for short term (1-2 weeks). Just prior to usage, add DTT to a final concentration of 1 mM.
• 1X Kinase/ATP/Substrate Solution: For each kinase assay, freshly prepare 50 μL of 1X Kinase/ATP/Substrate Solution by adding 1 μL of 10 mM ATP solution, 2 μL of ROCK substrate to 47 μL of 1X Kinase Buffer containing DTT. 4

Testdurchführung:

I. Kinase Reaction 1a. For Immunoprecipitations with anti-ROCK antibody: ROCK is first immunoprecipitated from cell or tissue lysate with anti-ROCK antibody and Protein A/G bead slurry. Immediately before kinase assay, wash bead slurry once with 1X Kinase Buffer, remove all supernatant, assay immediately by adding 50 μL of 1X Kinase/ATP/Substrate Solution directly to the beads and mixing well. 1b. Purified Kinase or Cell Lysate: Purified kinase or cell lysate sample can be used directly in the kinase assay or further diluted with 1X Kinase Buffer. Add 25 μL of ROCK sample to a microcentrifuge tube, and initiate kinase reaction by adding 50 μL of 1X Kinase/ATP/Substrate Solution to the ROCK sample.

(optional) Add 2 μL of the provided active ROCK-II and 23 μL of 1X Kinase Buffer to a microcentrifuge tube, initiate kinase reaction by adding 50 μL of 1X Kinase/ATP/Substrate Solution.

Incubate the tubes at 30 °C for 30-60 minutes with gentle agitation.

Stop kinase reaction by adding 25 μL of 4X reducing SDS-PAGE sample buffer.

Boil each sample for 5 minutes.

Centrifuge each sample for 10 seconds at 12,000 x g. II. Electrophoresis and Transfer

1. Load 20 μL of supernatant to a polyacrylamide gel. Also, it's recommended to include a pre- stained MW standard (as an indicator of a successful transfer in step 3).
2. Perform SDS-PAGE as per the manufacturer's instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane as per the manufacturer's instructions.

III. Immunoblotting and Detection (all steps are at room temperature, with agitation)

1. Following the electroblotting step, immerse the PVDF membrane in 100 % Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes. Note: If Nitrocellulose is used instead of PVDF, this step should be skipped.
2. Block the membrane with 5 % non-fat dry milk in TBST for 1 hr at room temperature with constant agitation.
3. Incubate the membrane with the anti-phospho-MYPT1 (Thr696) antibody, freshly diluted 1:1000 in 5 % non-fat dry milk/TBST, for 2 hr at room temperature with constant agitation. Note: To conserve antibody, incubations should be performed in a plastic bag.
4. Wash the blotted membrane three times with TBST, 5 minutes each time.
5. Incubate the membrane with the secondary antibody, HRP-conjugated, freshly diluted in 1:1000 in 5 % non-fat dry milk/TBST, for 1 hr at room temperature with constant agitation. 5
6. Wash the blotted membrane three times with TBST, 5 minutes each time.
7. Use the detection method of your choice. We recommend enhanced chemiluminescence reagents from Pierce.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid multiple freeze/thaw cycles.

Lagerung: -20 °C/-80 °C

Informationen zur Lagerung: Store active ROCK-II at -80°C and all other kit components at -20°C. Avoid multiple freeze/thaw cycles.

Referenzen

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Georgess, Mazzorana, Terrado, Delprat, Chamot, Guasch, Pérez-Roger, Jurdic, Machuca-Gayet: "Comparative transcriptomics reveals RhoE as a novel regulator of actin dynamics in bone-resorbing osteoclasts." in: Molecular biology of the cell, Vol. 25, Issue 3, pp. 380-96, (2014) (PubMed).

Chandrasekharan, Dechert, Davidson, Waitkus, Mavrakis, Lyons, Beach, Li, Egelhoff, Fox, DiCorleto: "Release of nonmuscle myosin II from the cytosolic domain of tumor necrosis factor receptor 2 is required for target gene expression." in: Science signaling, Vol. 6, Issue 284, pp. ra60, (2013) (PubMed).

Gupta, Davis, Nelson, Young, Alkayed: "Soluble epoxide hydrolase: sex differences and role in endothelial cell survival." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 32, Issue 8, pp. 1936-42, (2012) (PubMed).

González-Forero, Montero, García-Morales, Domínguez, Gómez-Pérez, García-Verdugo, Moreno-López: "Endogenous Rho-kinase signaling maintains synaptic strength by stabilizing the size of the readily releasable pool of synaptic vesicles." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 32, Issue 1, pp. 68-84, (2012) (PubMed).

Wang, Shi, Xie, Guo, Chen: "Response gene to complement 32 promotes vascular lesion formation through stimulation of smooth muscle cell proliferation and migration." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 31, Issue 8, pp. e19-26, (2011) (PubMed).

Xiao, Eto, Kazanietz: "ROCK mediates phorbol ester-induced apoptosis in prostate cancer cells via p21Cip1 up-regulation and JNK." in: The Journal of biological chemistry, Vol. 284, Issue 43, pp. 29365-75, (2009) (PubMed).

Li, Chang, Chiao, Kang, Xia, Zhu, Fleming, Evans, Chiao: "TrkBT1 induces liver metastasis of pancreatic cancer cells by sequestering Rho GDP dissociation inhibitor and promoting RhoA activation." in: Cancer research, Vol. 69, Issue 19, pp. 7851-9, (2009) (PubMed).

Antigendetails

Hintergrund: Members of the Rho family are essential regulatory components of the signaling pathway that direct cell motility, adhesion, and cytokinesis through reorganization of actin cytoskeleton. Rho is activated by extracellular signals such as lysophosphatidic acid (LPA). The actions of Rho are mediated by downstream Rho effectors. One of these effectors is Rho-associated kinase (ROCK). Two ROCK isoforms have been identified: ROCK-I (also known as ROKβ) and ROCK-II (also known as Rho Kinase and ROKα). ROCK mediates Rho signaling and reorganizes actin cytoskeleton through phosphorylation of several substrates that contribute to the assembly of actin filaments and contractility. For example, ROCK inactivates myosin phosphatase through the specific phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr696, which results in an increase in the phosphorylated content of the 20- kDa myosin light chain (MLC20).