Rat ROCK1(Rhoassociated, coiled-coil containing) ELISA Kit

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SKU:
RTFI01306
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q63644
Sensitivity:
75pg/ml
Range:
125-8000pg/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
p160 ROCK 1, P160ROCK, PRO0435, ROCK1
Reactivity:
Rat
Frequently bought together:

Description

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Product Name:Rat ROCK1 (Rhoassociated, coiled-coil containing) ELISA Kit
Product Code:RTFI01306
Size:96 Assays
Target:Rat ROCK1
Alias:p160 ROCK 1, P160ROCK, PRO0435, ROCK1
Reactivity:Rat
Detection Method:Sandwich ELISA, Double Antibody
Sensitivity:75pg/ml
Range:125-8000pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Rat ROCK1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat ROCK1 in samples. Please contact us for more information.
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat ROCK1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Please get in contact for more information.
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:Q63644
UniProt Protein Function:ROCK1: Protein kinase which is a key regulator of actin cytoskeleton and cell polarity. Involved in regulation of smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and motility via phosphorylation of DAPK3, GFAP, LIMK1, LIMK2, MYL9/MLC2, PFN1 and PPP1R12A. Phosphorylates FHOD1 and acts synergistically with it to promote SRC-dependent non-apoptotic plasma membrane blebbing. Phosphorylates JIP3 and regulates the recruitment of JNK to JIP3 upon UVB-induced stress. Acts as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability. Acts as a negative regulator of VEGF-induced angiogenic endothelial cell activation. Required for centrosome positioning and centrosome-dependent exit from mitosis. Plays a role in terminal erythroid differentiation. May regulate closure of the eyelids and ventral body wall by inducing the assembly of actomyosin bundles. Promotes keratinocyte terminal differentiation. Homodimer. Interacts with RHOB, RHOC, MYLC2B snd PTEN. Interacts with RHOA (activated by GTP), CHORDC1, DAPK3, GEM, JIP3, RHOE, PPP1R12A, PFN1, LIMK1, LIMK2 and TSG101. Interacts with FHOD1 in a Src-dependent manner. Detected in blood platelets. Activated by RHOA binding. Inhibited by Y- 27632. Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family.
UniProt Protein Details:

Protein type:AGC group; DMPK family; EC 2.7.11.1; Kinase, protein; Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor); ROCK subfamily

Chromosomal Location of Human Ortholog: 18p13

Cellular Component: cytoskeleton; lamellipodium; plasma membrane; ruffle

Molecular Function:GTP-Rho binding; protein serine/threonine kinase activity

Biological Process: actin cytoskeleton organization and biogenesis; bleb formation; cortical actin cytoskeleton organization and biogenesis; cytoskeleton organization and biogenesis; I-kappaB kinase/NF-kappaB cascade; leukocyte adhesion; leukocyte migration; leukocyte tethering or rolling; membrane to membrane docking; myoblast migration; negative regulation of angiogenesis; negative regulation of neuron apoptosis; negative regulation of protein binding; positive regulation of focal adhesion formation; regulation of actin cytoskeleton organization and biogenesis; regulation of actin filament-based process; regulation of keratinocyte differentiation; Rho protein signal transduction

NCBI Summary:protein serine/threonine kinase; involved in intracellular signaling [RGD, Feb 2006]
UniProt Code:Q63644
NCBI GenInfo Identifier:47605939
NCBI Gene ID:81762
NCBI Accession:Q63644.1
UniProt Secondary Accession:Q63644,Q7TP31,
UniProt Related Accession:Q63644
Molecular Weight:114,243 Da
NCBI Full Name:Rho-associated protein kinase 1
NCBI Synonym Full Names:Rho-associated coiled-coil containing protein kinase 1
NCBI Official Symbol:Rock1  
NCBI Official Synonym Symbols:ROCK-I; P160Rock  
NCBI Protein Information:rho-associated protein kinase 1
UniProt Protein Name:Rho-associated protein kinase 1
UniProt Synonym Protein Names:Liver regeneration-related protein LRRG199; Rho-associated, coiled-coil-containing protein kinase 1; Rho-associated, coiled-coil-containing protein kinase I; ROCK-I; p150 RhoA-binding kinase ROK beta; p160 ROCK-1; p160ROCK
Protein Family:Rho-associated protein kinase
UniProt Gene Name:Rock1  
StepProcedure
1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37°C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol
Serum:

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.

Plasma: Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge samples at 4°C for 15 mins at 1000 — g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid: Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant: Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell Lysates: Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or-80°C.
Tissue Lysates: Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk: Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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