CY-1160
Contents
1. y 719312x 119 59 R 0 9972 Em for ATP 4 5 uM Cat CY 1160 14 Version 140318 Rho kinase Assay Kit f Aolex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 2 Km for ATP recombinant DMPK y 7 38 084r 88 500 F 0 9945 Em for ATF 2 3 uM Fig 4 1 Effect of Rho kinase specific inhibitor Y27632 on activity of recombinant ROCK II catalytic domain 1040 120nM Istenzitz 95 of control 1 0 10 0 Y27632 vM Cat CY 1160 15 Versions 140318 Rho kinase Assay Kit v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures AN Fig 4 2 Effect of Rho kinase specific inhibitor Y27632 on activity of recombinant DMPK Relative intensity of control 0 20 40 80 100 Y 27632 uM Fig 5 RESOURCE Q column elution profile of Rho activity Rabbit Brain crude extract E ET Da T 0 T rae T Aa te odes 0 2 4 6 8 10 12 14 15 18 20 22 24 26 28 30 Fraction Number C CY 1160 16 Versions 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 6 Measurment of Rho kinase II activity in cell extract of 293T which had been transfected with Rho kinase II catalytic domain expression plasmid DNA 1 0 oo Oe 07 M 5 4 a3 a2 1 a 0 0001 0 001 0 01 0 1 cell extract ul reaction vol PROCKI CD r uM Y27633 ROCKI CD 10ull Y2762. 1 Following the above table add the Reagents to each wellgof the microplate Finally initiate the reaction by adding 10 uL of Your enzyme fraction or Buffer to each well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 30 for 30 minutes 2 Follow the Standard Assay steps 5 10 page 6 7 Evaluation of Results 1 Average the absorbance values for the Rho kinase sample duplicates positive control and all experimental sample duplicate values when applicable When the Rho kinase II positive control 10 m units assay is included as an internal control for the phosphorylation reaction the absorbance value should be greater than 1 0 with a background Jess than 0 15 2 For screening of purification chromatography fractions on graph paper plot the mean absorbance values for each of the samples on the Y axis versus the fraction number on the X axis to determine the location of the eluted purified Rho kinase 3 For kinetic analysis on graph paper plot the mean absorbance values for each of the time points on the Y axis versus the time of each reaction minutes on the X axis Assay Charact fistics The CycLex ResearehzProduct Rho kinase Assay Kit has been shown to detect the activity of Rho kinase family in column fractions The assay shows good linearity of sample response The assay may be used to follow the purification of Rho kinases or may be used to detect the presence of Rho kinases in c l
3. 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and standards should be assayed in duplicate The following components are supplied and are sufficient for the one 96 well microtiter plate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated with recombinant MBS C terminus 654 880 a a as Rho kinase substrate 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Kinase Buffer One bottle containing 20 mL of 1X buffer used for Kinase Reaction Buffer and sample dilution 20X ATP One vial of lyophilized ATP Na salt HRP conjugated Detection Antibody One vial containing 12 mL of ARP horseradish peroxidase conjugated anti phospho MBS T696 AF 20 antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2SO Ready to se Materials Required but not Provid d Recombinant Rho kinase II positive control Ayailable from CycLex Cat CY E1160 1 The positive control should be added to the first well at 10 M units well Recombinant DMPK positive control Available from CycLex Cat CY E1160 2 The positive control should be added to the first well at I0 munits well 10X Y 27632 100
4. Product Rho kinase Assay Kit have been tested for stability Reagents should not be used beyond the stated xpiration date Upon receipt kit reagents should be stored at 4 C except the ATP must be stored at 20 G Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing d desiccant pack For research use only not for use in diagnostic r therapeutic procedures Cat CY 1160 10 Version 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Numerous extraction and purification methods can be used to isolate Rho kinases The following protocols have been shown to work with a number of different tissues and enzyme sources and are provided as examples of suitable methods Crude samples can frequently be used without dilution while more concentrated or highly purified Rho kinases should be diluted It is strongly advised that the user always perform an initial experiment to determine the proper dilution to be used in subsequent experiments This need not be any more than a single time point assay using serial dilutions of the crude extract cell lysate or sample fraction taken prior to a purification step One eight well strip of the substrate plate should be sufficient for this initial experiment All sample preparation should be performed at 4 C and recovered fractions should be kept at 4 C to prevent loss of enzyma
5. TA Identification of the endogenous smooth muscle myosin phosphatase associated kinase Proc Natl Acad Sci USA 98 5 2419 24 2001 9 Niiro N and Ikebe M Zipper interacting protein kinase induces Ca 2 free smooth muscle contraction via myosin light chain phosphorylation J Biol Chem 276 31 29567 74 2001 10 Kimura K Ito M Amano M Chihara K Fukata Y Nakafuku M Yamamori B Feng J Nakano T Okawa K Iwamatsu A Kaibuchi K Regulation of myosin phosphatase by Rho afid Rho associated kinase Rho kinase Science 273 5272 245 8 1996 11 Kawano Y Fukata Y Oshiro N Amano M Nakamura T Ito M Matsumura F 4nagaki M Kaibuchi K Phosphorylation of myosin binding subunit MBS of myosin phosphatase by Rho kinase in vivo J Cell Biol 147 5 1023 38 1999 12 Nagumo H Sasaki Y Ono Y Okamoto H Seto M Takuwa Y Rho kipase inhibitor HA 1077 prevents Rho mediated myosin phosphatase inhibition in smooth musele cells Am J Physiol Cell Physiol 278 1 C57 65 2000 13 Matsui T Amano M Yamamoto T Chihara K Nakafuku M Ito M Nakano T Okawa K Iwamatsu A Kaibuchi K Rho associated kinase a novel serine threonine kinase as a putative target for small GTP binding protein Rho EMBO J 15 9 2208 16 1996 Related Products Rho kinase Positive control Cat CY E1160 1 DMPK Positive control Cat CY E1160 2 Anti phospho MBS MYPT1 Thr696 mongelonal antibody AF 20 Cat CY M1011 PRODUCED BY CycLex Co Ltd
6. a horseradish pefOxidase conjugate of AF20 a anti phospho MBS threonine 696 specific antibody which then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TMB from a colorless solution fo a blue solution or yellow after the addition of stopping reagent The color is quantified by Spectrophotometry and reflects the relative amount of Rho kinase activity in the sample For kinetic analysis the sample containing Rho kinase is added to the wells in a similar fashion and at varying times the reaction is stopped by the addition of a chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as before The CycLex Research Product CycLex Rho kinase Assay Kitis designe d to accurately determine the presence and relative amount of Rho kinase activity in purification eolumn fractions and to determine non isotopic kinetic analysis of Rho kinase activity Car ful attention to extraction methods and the assay protocol will provide the investigator with a reliable tool for the evaluation of Rho kinases Summary of Procedure Add 100 uL of sample to the wells 4 Incubate for 30 min at 30 C Wash the wells Y Add 100 WL of ARP conjugated anti phospho specific antibody Incubate for 30 min at room temp Wash the wells t Add 100 uL of Substrate Reagent Add 100 uL of Stop Solution t Measure absorbance at 450 nm Cat CY 1160 3 Version 140318 Rho kinase Assay Kit
7. activity is enriched in the 0 3 M NaCl eluent 6 Dilute the pooled Rho kinase containing fraction with three volumes of S buffer 25 mM MES NaOH pH 6 0 0 05 Triton X4 00 0 55mM EDTA 1 mM EGTA 1 ug mL pepstatin 0 5 ug mL leupeptin 2 mM NaF 2 mM Na3VO 5 mM beta mercaptoethanol 5 glycerol 7 Apply the diluted sample4o a 1 x 2 cm column of S Sepharose Amersham Pharmacia Biotech that has been equilibrated with S buffer containing 50 mM NaCl 8 Wash the column with ten column volumes of S buffer containing 50 mM NaCl 9 Elute the protein with a linear gradient of NaCl 0 05 0 6 M in S buffer collecting 1 mL fractions Th se samples are now ready for analysis according to the instructions provided in the Detailed Protocol Collect the Rho kinase containing fraction Usually Rho kinase activity is enriched in the 0 2 M NaCl eluent Cat CY 1160 11 Version 140318 Rho kinase Assay Kit 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Cell Culture Lysates Harvest and pellet cells by centrifugation using standard methods 2 Resuspend the cell pellet with an appropriate extraction buffer for example 50 mM Tris HCl pH 8 0 0 1 Triton X 100 1 mM EDTA 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 1g mL leupeptin 2 mM NaF 2 mM Na VO 10 mM beta mercaptoethanol and lyse the resuspended cells using either a Dounce Homogenizer sonication or three cycles of freezi
8. 0 550 or 450 595 nm can also be used Read the plate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Recommendations Special considerations when screening activators and inhibitors In order to estimate the inhibitory effect on Rho kinase family activity in the test chemicals correctly it is necessary to conduct the control experiment of Solvent control at least once for every experiment and Inhibitor control at least once for the first experiment in addition to Test sample as indicated in the following table When test chemicals cause an inhibitory effect on Rho kinase activity the level of A450 is weakened as compared with Solvent control The high level of A450 is not observed in Inhibitor control usually A450 lt 0 2 Are eaS Test Solvent Inhibitor y reag sample control control Kinase Reaction Buffer 80 uL 80 uL 80 uL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10 uL 10X Y 27632 100 uM 10 uL CycLex Rho kinase Positive Control Pm unit uL 10 uL 10 uL 10 uL or your enzyme fraction 10X Y 27632 See page 4 section Materials Required but not Provided Cat CY E1160 1 See page A section Materials Required but not Provided 1 Following the above tablejadd the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of Diluted CycLex Rho kinase to eac
9. 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyelex co4p URL http wwwWw eyclex eo jp CycLex Circulex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 1160 18 Version 140318
10. 32 References p Ishizaki T Mackawa M Fujisawa K Okawa K Iwamatsu A Fujita A Watanabe N Saito Y Kakisuka A Morii N and Narumiya SBE MBO J 15 1885 1893 1996 2 Fujisawa K Fujita A Ishizaki T S ito Y and Narumiya S J Biol Chem 271 23022 23028 1996 3 Nakamura M Nagano T Chikam T and Nishida T Invest Ophthal Vis Sci 42 941 947 2001 4 Rao P V Deng P F Kumail J and Epstein D L Invest Ophthal Vis Sci 42 1029 1037 2001 n Muranyi A Zhang R Li F Hirano K Ito M Epstein HF Hartshorne DJ Myotonic dystrophy protein kinase ph sphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity WEBS Lett 493 2 3 80 4 2001 6 Koyama M Ito M Feng J Seko T Shiraki K Takase K Hartshorne DJ Nakano T Phosphorylation of CPI 17 an inhibitory phosphoprotein of smooth muscle myosin phosphatase by Rho kinase FEB Sebett 473 3 197 200 2000 N Hamaguchi Ito M Feng J Seko T Koyama M Machida H Takase K Amano M Kaibuchi K Hartshorne DJ Nakano T Phosphorylation of CPI 17 an inhibitor of myosin phosphatase by protein Cat CY 1160 17 Version 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures kinase N Biochem Biophys Res Commun 274 3 825 30 2000 8 MacDonald JA Borman MA Muranyi A Somlyo AV Hartshorne DJ Haystead
11. P Rho kinase Assay Kit Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring Rho kinase and DMPK Activity CycLex Rho kinase Assay Kit Cat CY 1160 Intended Use essssseseeee 1 s lr M 1 TintroductiOn cccceceescceeseseeeceeneeceesteeeessnees 2 Principle of the Assay 3 Materials Provided ssssse 4 Materials Required but not Provided 4 Precautions and Recommendations 5 Detailed Protocol esses 6 9 Evaluation of Results sssss 9 Assay Characteristics 9 Troubleshooting iisuseee seen tran tna toria 10 Reagent SEDIT siente ert arta epet ineo 10 Sample Preparation esses reino seite 11 12 Example of Test Reshlts s serre 13 47 References essere 17 18 Related Products esses T8 Intended Use The CycLex Research Product CycLex Rho kinase Assay Kit is primarily designed to measure the activities of purified Rho kinase or DMPK f r the rapid and sensitive evaluation of inhibitors or activators The phospho specific monoclofial antibody used in this assay kit has been demonstrated to recognize the phospho threonine 696 residue in MBS MYPTI which is phosphorylated by Rho kinase or DMPK Myotonic dystrophy protein kinase family kinases Additionally column fractio
12. ayed in duplicate Uo To assay individual col mn fractions add 10 uL of each fraction to the wells of the assay plate on ice Crude lysates or ell extracts should be added to wells either neat or diluted as describe above with Kinase Buffer if necessary Suggested starting dilutions are 1 5 1 10 and 1 20 Duplicate wells containing TO mUnits 10 uL Rho kinase II positive control Cat CY E1160 1 should be included in each assay as a positive control for phosphorylation 4 Begin the kinase reaction by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer and incubate at 30 C for 30 minutes 5 Wash wells five times with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration Cat CY 1160 6 Version 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures 6 Pipette 100 uL of HRP conjugated Detection Antibody into each well cover with a plate sealer and incubate at room temperature ca 25 C for 30 minutes Discard any unused conjugate 7 Wash wells five times as same as in step5 oo Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 5 15 minutes 9 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 10 Measure absorbance in each well using a spectrophotometric plate reader a
13. ental conditions may vary an aliquot of the Rho kinase II positive control Cat CY E1160 1 available separately from CycLex should be included in each assay as a positive control Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solution 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer provided to 900 mL of ddH5O Mix well Store at 4 C for two weeks or 20 C for long terdi storage 2 Prepare 20X ATP Solution by adding 0 8 mL of ddH O to the vial of 20X ATP provided lyophilized Mix gently until dissolved The final concentration of the 20X A TP Solution should be 2 5 mM Store the solution in small aliquots e g 100 uL at 20 C 3 Prepare Kinase Reaction Buffer by mixing following reagents 96 assays 10 assays 1 assay Kinase Buffer provided 9 5 mL 950 pL 95 pL 20X ATP Solution 0 5 mL 50 pL 5 uL Total 10 mL 1000 uL 100 uL You will need 80 90 uL of Kinase Reaction Buffer per assay well Mix well Discard any unused Kinase Reaction Buffer after use Standard Assay 1 Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Prepare all samples diluted with Kinase Buffer as needed All samples should be ass
14. g domain of ROCKI to a region between residues 934 1015 ROCK2 Rho kinase alpha is a serine threonine kinase that regulates cytokinesis smooth muscle contraction the formation of actin stress fibers and focal adhesions and the activation of the c fos serum response element ROCK2 which is an isozyme of ROCK1 is a target for the small GTPase Rho Nakamura et al 2001 studied the role of Rho in the migration of corneal epithelial cells rabbit They detected both ROCK1 and ROCK2 in the corneal epithelium at protein and mRNA levels They found that exoenzyme C3 a Rho inhibitor inhibits corneal epithelial migration in a dose dependent manner and prevents the stimulatory effect of the Rho activator lysophosphatidic acid KPA Both cytochalasin B an inhibitor of actin filament assembly and ML7 an inhibitor of myosin light chain kinase also prevent LPA stimulation of epithelial migration The authors suggested that Rho mediates corneal epithelial migration in response to external stimuli by regulating the organization of the actin cytoskeleton Rao et al 2001 investigated the role of Rho kinase in the4modulation of aqueous humor outflow facility The treatment of human trabecular meshwork and canal of Schlemm cells with a Rho kinase specific inhibitor led to significant but reversible changes in cell shape and decreased actin stress fibers focal adhesions and protein phosphotyfdsine staining Based on the Rho kinase inhibitor induced cha
15. h well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 30 C for 30 minutes 2 Follow the Standard Assay steps 5 10 page 6 7 Cat CY 1160 Version 140318 Rho kinase Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures 4 YCLEX Special considerations when measuring precise Rho kinase activity In order to measure the activity of Rho kinase family correctly it is necessary to conduct the control experiment of Inhibitor control at least once for every experiment and ATP minus control at least once for the first experiment in addition to No enzyme control as indicated in the following table Although the level of A450 increases in Test sample when Rho kinase family enzyme activity isimthe sample the high level of A450 is not observed in Inhibitor control ATP minus contg l and No enzyme control Assay reng nts Test Sample Inhibitor ATP minus Positive Noyenzyme control control control control Kinase Reaction Buffer 90 uL 80 uL 90 E 90 uL Kinase Buffer provided 90 uL 10X Y 27632 100 uM 10 pL Your enzyme fraction 10 pL 10 pL 10 uL CycLex Rho kinase Positive g J 1L Control 1 m unit uL Buffer 10 uL See page 4 section Materials Required but not Provided Cat CY E1160 1 See page 4 section Materials Required but not Provided
16. l lysates It should be noted that this assay kit detects not only Rho kinase activity but also other protein kinase activities e g ZIP kinase DMK and ILK in crude extract and column sample in the absence of Rho kinase inhibitor 140318 Cat CY 1160 9 Version Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 The Rho kinase positive control should be run in duplicate using the protocol described ingthe Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 The reaction curve is nearly a straight line if the kinetics of the assay is of the first order Variations in the protocol can lead to non linearity of the curve as can assay kinetics that are other than first order For a non linear curve point to point or quadratic curve fit methods should be used 3 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 4 Overall low signal may indicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add S bstrate Reagent immediately after wash Reagent Stability All of the reagents included in the CycLex Research
17. monoclonal antibody as a reporter molecule in a 96 well ELISA format This assay provides a non isotopic sensitive and specific method to detect Rho kinase family activity Cat CY 1160 2 Version 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex Rho kinase Assay Kit is a single site semi quantitative immunoassay for Rho kinase activity Plates are pre coated with a substrate corresponding to recombinant the C terminus of MBS Myosin Binding Subunit of myosin phosphatase which contains a threonine residue that may be phosphorylated by DMPK family members including Rho kinase ROCK1 and ROCK2 MRCK Myotonic Dystrophy kinase related Cdc42 binding Kinase and DMPK Myotonic Dystrophy Protein Kinase The detector antibody is AF20 an antibody that specifically detects only the phosphorylated form of threonine 696 on MBS The CycLex Research Product CycLex Rho kinase Assay Kit may be used to determine the presence of Rho kinase activity in purification column fractions or to follow the kinetics of a purified or partially purified Rho kinase protein as well as screening Rho kinase inhibitor To perform the test the sample is diluted in Kinase Buffet pipetted into the wells and allowed to phosphorylate the bound substrate in the presence of Met and ATP The amount of phosphorylated substrate is measured by binding it with
18. ng and thawing 3 Transfer extracts to microcentrifuge tubes and centrifuge for 5 minutes 4 Aliquot cleared lysate to a clean microfuge tube These samples are now ready for analysis according to the instructions provided in the Detailed Protocol NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS BEING INVESTIGATED AND MUST BE DETERMINED BY THE INDIVIDUAL USER NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED Example of Test Results Fig 1 1 Dose dependency of Rho kinase II catalytic domain enzyme reaction 0 0 0 0001 0 101 0 0L 11 l 10 100 1000 Rho kinase II mUnis Cat CY 1160 12 Version 140318 wry Rho kinase Assay Kit T7 Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 1 2 Dose dependency of recombinant DMPK enzyme reaction Q 1 0 A450 ue 0 5 0 0 0 5 10 15 20 DMPK mUnits gt zyme reaction Fig 2 1 Time course of recombinant Rho kinase II catalytic do 80 75 90 105 Reaction time min C CY 1160 13 Versions 140318 Rho kinase Assay Kit f Aolex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 2 2 Time course of recombinant DMPK enzyme reaction 20 40 8l all 100 120 Reaction time frain Fig 3 1 Km for ATP recombinant Rho kinase II catalytic domain
19. nges in myosin light chain phosphorylation and actomyosin organization the authors suggested that cellular relaxation and lossf cell substratum adhesions in the human trabecular meshwork and canal of Schlemm cells could result in ither increased paracellular fluid flow across the canal of Schlemm or altered flow pathway through the juxtacanalicular tissue thereby lowering resistance to outflow They suggested Rho kinase as a potential target for the development of drugs to modulate intraocular pressure in glaucoma patients Measurement of Rho kinase activity The protocol generally regarded as niost sensitive for the quantitative measurement of Rho kinase activity involves incubation of the Rho kinase sample with substrate either a natural or synthetic polypeptide such as Long S6 Kinase substrate peptide in the presence of Mg and P labeled ATP The reaction is terminated by spotting a sample onto a filter paper disc followed by immersion in acid to precipitate the radiolabeled product The filter papers are then washed extensively to remove unincorporated radiolabel fand the radioactivity is counted While sensitive this method is labor intensive generates fazardous radioactive waste and depends on a radioisotope of short half life It is particularly unsuitable When kinase assays are only performed on an infrequent basis The CycLex Research Product CyeLex Rho kinase Assay Kit uses a peroxidase coupled anti phospho MBS threonine696
20. ns of cultured primary cell cell line or tissue homogenate can be assayed for Rho kinase family activity with the CycLex Research Product CyeLex Rho kinase Assay Kit if the appropriate dose of Rho kinase specific inhibitor e g Y 27632 or H A 1077 is used Applications of this kit include 1 Monitoring the purification of Rho kinase or DMPK family kinase 2 Screening inhibitors or activators of Rho kinase or DMPK family kinase 3 Detecting theeffects of pharmacological agents on Rho kinase or DMPK family kinase This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage Upon r ceiptystore all components at 4 C Don t expose reagents to excessive light Cat CY 1160 1 Version 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction The small GTPase Rho regulates formation of focal adhesions and stress fibers of fibroblasts as well as adhesion and aggregation of platelets and lymphocytes by shuttling between the inactive GDP bound form and the active GTP bound form Rho is also essential in cytokinesis and plays_a role in transcriptional activation by serum response factor Ishizaki et al 1996 identified the protein serine threonine kinase ROCKI Rho kinase beta which they called p160 ROCK which is activated when bound to the GTP bound form of RhoA Fujisawa et al 1996 localized the Rho bindin
21. t dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good petformance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels Note 2 Reliable signals are obtained when either O D values do not xceed 0 25 units for the blank no enzyme control or 2 5 units for the Rho kinase positive control Note 3 If the microplate reader is not capable of reading absorbance greater than the absorbance of the Rho kinase II positive control perform a4 econd reading at 405 nm A new O D values measured at 405 nm is used to determine Rho kinase activity of off scale samples The readings at 405 nm should not replace the on cale readings at 450 nm Kinetic Assay 1 Remove the appropriate number of microtitef wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Prepare all samples diluted with Kinase Buffer as needed All samples should be assayed in duplicate Uo To assay individual column fractions add 10 uL of each fraction or Rho kinase II positive control Cat CY E1160 1 to the wells of the assay plate Duplicate wells con
22. taining 10 mUnits 10 uL Rho kinase II positive control Cat CY E1160 1 should be included in each assay as a positive control for phosphorylation 4 Begin kinase reaction by addition of 90 uL Kinase Reaction Buffer in duplicate per well in timed intervals suggested interval is 5 minutes but should be individually determined for each system After the final addition incubate at 30 C for 20 minutes 5 Stop the r action by flicking out the contents Alternatively the reaction may be terminated by the addition o 450 pL 0 1 M Na EDTA pH 8 0 to each well 6 Wash wells five times with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration Cat CY 1160 T Version 140318 yclex User s Manual Rho kinase Assay Kit For Research Use Only Not for use in diagnostic procedures 7 Pipette 100 uL of HRP conjugated Detection Antibody into each well cover with a plate sealer and incubate at room temperature ca 25 C for 30 minutes Discard any unused conjugate after use oo Wash wells as same as in Step 6 9 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 10 15 minutes 10 add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 11 Measure absorbance in each well using a spectrophotometric plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 45
23. tic activity CAUSION It should be noted that this assay kit detects not only Rho kinase activity but also other protein kinases e g ZIP kinase DMK and ILK in crude extract atid column sample You should trace Rho kinase protein level by western blotting in column fractions Column Purification Fractions 1 Homogenize 5 8 g of fresh tissues brain kidney platelet etc injfiour volumes of an appropriate extraction buffer for example 50 mM Tris HCl pH 8 0 0 1 oZEriton X 100 1 mM EDTA 1 mM EGTA 0 5 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin LOamM NaF 2 mM Na3VO 10 mM beta mercaptoethanol in a Potter Elvehjem tissue grinder 2 Centrifuge the homogenate for 30 min at 30 000 x g to pellet the insoluble membrane organelle fraction 3 Apply the supernatant fraction to a 1 x 8 cm column of Q Sepharose Amersham Pharmacia Biotech that has been equilibrated with Q buffer 20 mM Tris HCl pH 8 0 0 1 Triton X 100 0 5 mM EDTA 1 mM EGTA 1 pg mL pepstatin 0 5 ug mE leupeptin 5 mM beta glycerophosphate 2 mM NaF 2 mM Na3VO 5 mM beta mercaptoethanol containing 50 mM NaCl 4 Wash the column with five column volumes of Q buffer containing 50 mM NaCl 5 Elute the protein with a linear gradient of NaCl 0 05 0 6 M in Q buffer collecting 1 2 mL fractions These samples are now ready forganalysig according to the instructions provided in the Detailed Protocol Collect the Rho kinase containing fraction Usually Rho kinase
24. uM Y 27632 Calbiochem Cats 688001 5 mM solution diluted 1 50 in water Pipettors 2 20 uL 20 200 uL and 20041000 uE precision pipettors with disposable tips Precision repeating pipettor Wash bottle or multichannel dispenser for plate washing Microcentrifuge and tubes for sample preparation Vortex mixer e Plate reader capable of meas ring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength of 450m which will give a somewhat higher reading 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized watenof the highest quality Disposable paper towels Cat CY 1160 4 Version 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Store the ATP at 20 C in aliquots Store all other components at 4 C Do not expose reagentsato excessive light Avoid freeze thaw cycles Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock peuch Which must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indicated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residue from glassware Use deionized
25. water of the highest quality Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or other chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipet or ingest any of the reagents Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled Dispose of tetra methylbenzidine TMB containing sol tions in compliance with local regulations Avoid contact with Substrate Solution which contains hydrogen peroxide Avoid contact with Stop Solution which contains Sulfuric Acid n case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1160 5 Version 140318 Rho kinase Assay Kit 4 P Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Rho kinase Assay Kit is provided with removable strips of wells so the assay cangbe carried out on separate occasions using only the number of strips required for the particular determination Since experim
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