96-Well ROCK Activity Assay Kit

Catalog No. ABIN2345100

Product Details

Detection Method: Colorimetric

Application: Activity Assay (AcA)

Sample Type: Cell Samples, Tissue Lysate

Characteristics: 96-well ROCK Activity Assay Kit is an enzyme immunoassay developed for detection of the specific phosphorylation of MYPT1 at Thr696 by ROCK. A strip well microtiter plate is precoated with a recombinant MYPT1. After incubating the substrate wells with ROCK samples (such as purified kinase, cell or tissue lysate) the phosphorylated MYPT1 is detected by an anti-phospho- MYPT1 (Thr696) antibody (Figure 1). 96-well ROCK Activity Assay Kit provides a non-isotopic, sensitive and specific method to monitor ROCK activity using its physiological substrate, it can also be used in screening ROCK inhibitors. The kit has detection sensitivity limit of 200 pg of active ROCK-II. A recombinant active ROCK-II is also provided as a positive control. Each kit provides sufficient quantities to perform up to 96 assays. Related Products 1. STA-415: ROCK Activity Immunoblot 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

Components:

1. ROCK Substrate Coated Plate : One strip well 96-well plate precoated with recombinant MYPT1
2. 10X Kinase Buffer : One 20 mL bottle of 250 mM Tris, pH 7.5, 100 mM MgCl2, 50 mM Glycerol-2-Phsophate, 1 mM Na3VO4
3. ATP Solution : One 100 μL vial Of 100 mM ATP
4. Anti-phospho-MYPT1 (Thr696) : One 20 μL vial
5. Secondary Antibody, HRP Conjugate : One 20 μL vial
6. Assay Diluent : One 50 mL bottle.
7. 10X Wash Buffer : One 100 mL bottle
8. Substrate Solution : One 12 mL amber bottle
9. Stop Solution (Part. No. 310808): One 12 mL bottle

Box 2 (shipped on blue ice packs)

Material not included:

1. ROCK sample (purified kinase, cell or tissue lysate)
2. Lysis Buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM 2-glycerophosphate, 1 % Triton X-100 or 1 % Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4 and Proteinase inhibitors.
3. DTT
4. 0.5 M EDTA
5. 30 °C incubator or water bath
6. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
7. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
8. Multichannel micropipette reservoir
9. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Safe non-radioactive ELISA format
• Active ROCK-II included as positive control

Reagent Preparation:

• 10X Kinase Reaction Buffer containing DTT and ATP: Just prior to usage, add DTT to a final concentration of 10 mM and ATP to a final concentration of 2 mM to the 10X Kinase Buffer. For Example, add 10 μL of 1M DTT (not provided) and 20 μL of 100 mM ATP solution to 970 μLof 10X Kinase Buffer. 10X Kinase Reaction Buffer containing DTT and ATP may be stored at 4 °C for short term (1-2 weeks).
• Diluted Active ROCK-II Postive Control: Just prior to usage, dilute the provided active ROCK-II (0.5 μg/mL) to 0.02 μg/mL with 1X Kinase Buffer. For example, add 8 μL of the active ROCK-II and 20 μL of 10X Kinase Buffer to 172 μL deionized water.
• 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
• Anti-Phospho-MYPT1 (Thr696) Antibody and HRP-Conjugated Secondary Antibody: Immediately before use dilute the anti-phospho-MYPT1 (Thr696) antibody 1:1000 and HRP-conjugated secondary antibody 1:1000 with Assay Diluent. Do not store diluted solutions.

Assay Procedure:

1. Purified kinase or cell lysate sample can be used directly in the kinase assay or further diluted with 1X Kinase Buffer. Each sample should be assayed in duplicate.
2. Add 90 μL of the diluted active ROCK-II positive control or unknown ROCK samples to the wells of the substrate plate.
3. Initiate the kinase reaction by adding 10 μL of the 10X Kinase Reaction Buffer containing DTT and ATP. Mix well.
4. Cover with a plate cover and incubate the wells at 30 °C for 30-60 minutes with gentle agitation.
5. Stop kinase reaction by flicking out the content or by adding 50 μL of 0.5 M EDTA, pH 8.0, to each well.
6. Remove plate cover and empty wells. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
7. Add 100 μL of the diluted anti-phospho-MYPT1 (Thr696) antibody to each well.
8. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
9. Remove plate cover and empty wells. Wash the strip wells 3 times according to step 6 above.
10. Add 100 μL of the diluted HRP-conjugated secondary antibody to each well.
11. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker. 5
12. Remove plate cover and empty wells. Wash microwell strips 3 times according to step 6 above. Proceed immediately to the next step.
13. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature for 5-20 minutes on an orbital shaker.
14. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
15. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: -20 °C/-80 °C

Storage Comment: Store active ROCK-II at -80°C, ATP Solution at -20°C and all other kit components at 4°C. Avoid multiple freeze/thaw cycles.

References

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Target Details

Background: 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).