Home

CY-1173

1. Cat CY 1173 3 Version 150310 Pae CaM kinase II Assay Kit ca ycLex 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 supplie 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 bag with a desiccant pack Wells are coated with Syntide 2 as a 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 Oj and sample dilution 50X CaCl One vial containing 0 4 mL of 125 mM CaCl used for Kinase Buffer Ca CaM plus action Buffer Ca CaM minus 20X ATP One vial of lyophilized ATP Naz salt Q HRP conjugated Detection Antibody One vial containin A P horseradish peroxidase conjugated anti phospho Syntide 2 monoclonal antibody M Ready to use Substrate Reagent One bottle containing 20 mL Y substrate tetra methylbenzidine TMB Ready to use 50X EGTA One vial containing 0 4 mL of 100 mM EGTA used for Ki se R Stop Solution One bottle containing 20 mL of 1 N H2804 Ready to use C CY 1173 4 Version 150310 Pae CaM kinase II Assay Kit ca NycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provid
2. USING THESE PROCEDURES IS MADE OR IMPLIED C CY 1173 13 Version 150310 CaM kinase II Assay Kit yy s cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of CaM kinase II enzyme reaction 3 5 3 0 2 5 gt 2 0 lt lt 1 5 200 ng ml Calumodulin 1o 2 mM CaCl 0 5 0 ng ml Calumodulin i 0 mM CaCl 0 0 0 5 10 15 20 25 30 35 40 CamKII positive control m units Y Fig 2 Time course of recombinant CaM kinase II ope 3 5 3 0 2 5 2 0 OD450 1 5 1 0 0 5 0 0 0 15 30 45 Q Reaction Time min C CY 1173 14 Version 150310 CaM kinase II Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Calmodulin dependent activity of CaM kinase II 3 5 N 3 0 2 5 2 2 0 LO tis 1 0 0 5 0 0 0 100 150 200 ons dulin ng ml Q v Fig 4 Calcium dependent activity of CaM kinase O 3 0 2 5 2 0 1 5 A450 1 0 0 5 0 0 200 400 600 800 1000 Ca2 conc uM Q C CY 1173 15 Version 150310 CaM kinase II Assay Kit Ca NycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of EGTA on activity of recombinant CaM kinase II presence of 2 mM calcium and 200 ng m
3. 3 _ 10 uL i Control 1 5 m unit uL u Kinase Reaction Buffer Ca CaM plus and Ca CaM minus See page 7 section Preparation of Working Solution 10X Staurosporine V uUM aS e page 5 section Materials Required but not Provided xxx Cat CY E1173 See page 5 section Materials Required but not Provided 1 Following the above table add the Reagents to each well of 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 C for 30 minutes 2 Follow the Standard Assay steps 5 10 page 8 Cat CY 1173 10 Version 150310 aR CaM kinase II Assay Kit ory P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results 1 Average the absorbance values for the CaM kinase II sample duplicates positive control andgall experimental sample duplicate values when applicable When the CaM kinase II Positive Control 15 m units assay is included as an internal control for the phosphorylation reaction the absorbance y lue should be greater than 1 0 with a background less than 0 2 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 C
4. Sulfuric_Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1173 6 Version 150310 aR CaM kinase II Assay Kit ory P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CycLex CaM kinase II Assay Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the CaM kinase II Positive Control Cat CY E1173 including calmodulin available separately from CyeLex 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 ddH20 Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH20 to the vial of 20X ATP provided lyophilized Mix gently until dissolved The final concentration of the20X ATP Solution should be 1 25 mM Store the solution in small aliquots e g 100 uL at 20 C 3 Prepare Kinase Reaction Buffer Ca CaM plus by mixing following reagents 96 assays 10 assa
5. 4 C and recovered fractions should be kept at 4 C to prevent loss of enzymatic activity CAUSION It should be noted that this assay kit detects not only CaM kinase I attivity but also other protein kinase activities in crude extract and column sample You should trace CaM kinase II protein level by western blotting in column fractions Preparation of bovine brain extract 1 Homogenize fresh 10 15 g of cerebellum in three volumes of ice cold extraction buffer 100 mM PIPES pH 6 9 10 mM EDTA 10 mM EGTA 0 3 mM PMSB lyprg mL pepstatin 0 5 pg mL leupeptin 5 mM glycerophosphate 2 mM NaF 2 mM N amp VO4 20 mM 6 mercaptoethanol in a Potter Elvehjem tissue homogenizer 2 Centrifuge the homogenate for 20 min at 20 000 x amp to pellet the insoluble membrane organelle fraction Column Purification of CaM kinase II 3 Dilute resultant supernatant by addinges2 volumes of ice cold H2O containing 10 mM B mercaptoethanol 4 Load the supernatant onto a 2 x 10 cmolumn of phosphocellulose P11 Whatman equilibrated with Buffer A 30 mM Pipes pH 6 9 0 5 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 5 mM B glycerophosphate 2 mM NaF 2aaM Nagv O4 10 mM mercaptoethanol Nn Wash the column with six column volumes of Buffer A 6 Sequentially elute the protein with two column volumes of 150 mM NaCl in Buffer A and 350 mM NaCl in Buffer A Pool the lattem350 mM NaCl eluate for further purification of CaM kinase II 7 Add Ca
6. 96 well ELISA format This assay provides a non isotopic sensitive and specific method to detect CaM kinase IT activity The CycLex Research Product CycLex CaM kinase II Assay Kit is designed to accurately determine the presence and relative amount of CaM kinase II activity in purification column fractions and for the non isotopic kinetic analysis of CaM kinase II activity Careful attention to extraction methods and the assay protocol will provide the investigator with a reliable tool for the evaluation of CaM kinase II Cat CY 1173 2 Version 150310 aR CaM kinase II Assay Kit ory cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex CaM kinase II Assay Kit is a single site semi quantitative immunoassay for CaM kinase II activity Plates are pre coated with a newly designed Syntide 2 which can be efficiently phosphorylated by CaM kinase II on a microtiter plate The detector antibody is MS 6E6 an antibody that specifically detects only the phosphorylated Syntide 2 he Cyclex Research Product CycLex CaM kinase II Assay Kit might be used to determine the presence of CaM kinase II activity in purification column fractions or to follow the kinetics of a purified CaM kinase II protein as well as screening CaM kinase II inhibitor or activator To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed
7. Cl to the 350amM NaCl eluate at final concentration of 1 mM 8 Load the 350M NaCl eluate to a calmodulin Sepharose affinity column 1 x 4 cm 5 mg calmodulin 1 ml Sepharose equilibrated with Buffer B 40 mM Tris HCl pH 7 2 0 5 mM CaCl 1 mM _ dithiothreitol 0 2 mM PMSF 1 wug mL pepstatin 0 5 pg mL leupeptin 5 mM B glycerophosphate 2 mM NaF 2 mM Na3VOs containing 0 2 M NaCl 8 Wash the column with Buffer B containing 2 M NaCl until the absorbance at 280 nm reached baseline Cat CyY 1173 12 Version 150310 CaM kinase II Assay Kit A yCLex User s Manual For Research Use Only Not for use in diagnostic procedures A 9 Elute CaM kinase II with Buffer C 40 mM Tris HCl pH 7 5 2 5 mM EGTA 1 mM dithiothreitol 0 2 mM PMSF 1 pg mL pepstatin 0 5 ug mL leupeptin 5 mM glycerophosphate 2 mM NaF mM Na3VOa 10 Load the calmodulin Sepharose eluate onto MonoQ column 1 ml previously equilibrated with buffer D 20 mM Tris HCl pH 7 5 0 1 mM CaC12 1 mM dithiothreitol 0 2 mM P pepstatin 0 5 ug mL leupeptin 5 mM B glycerophosphate 11 Washed the column with 10 ml of buffer D containing 50 mM NaCl 12 Elute CaM kinase II with a linear NaCl gradient 0 05 0 4 M at 0 5 ml mi llecting 1 ml fractions NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS ELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY ING ON THE PARAMETERS BEING INVESTIGATED AND MUST B INED BY THE INDIVIDUAL USER NO WARRANTY OR GUARAN PERFORMANCE
8. aM kinase II 3 For kinetic analysis on graph paper plot the mean absorbance values for each ofthe time points on the Y axis versus the time of each reaction minutes on the X axis Assay Characteristics The CycLex Research Product CycLex CaM kinase II Assay Kit has been shown to detect the CaM kinase II activity in column fractions of human or animal cell lysates yThe assay shows good linearity of sample response The assay may be used to follow the purification offCaM kinase II Troubleshooting 1 The CaM kinase II positive control should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures jsignificantly 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 ean assay kinetics that are other than first order For a non linear curve point to point or quadratice 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 indicateahat desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do net allow the plate to dry out Add Substr
9. aR CaM kinase II Assay Kit ory aa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring CaM kinase II Activity CycLex CaM kinase II Assay Kit Cat CY 1173 Intended ISG ocanacarsaanteandadnietaieniecissamkectond 1 SOOO T 1 TiO WO My 2i153545 eccaede deve tevadsniecucanwletiee 2 Principle Of the Assay 3 Materials Provided s cisccsessscccsscsessssasvacesecsvess 4 Materials Required but not Provided 5 Precautions and Recommendation 6 Detailed Protocol ss scccccsivssvacceesssisiecececssveacaaes 7 10 Evaluation Of RESUlts ccccsccssdssceescessansvers 11 Assay Characteristics 11 Troubleshooting 5 5 csannzasbucevosssncssssannaeliureee 11 Reagent Stability 11 Sample Preparation seeseeeeeeeeeeeeeereereee 12 13 Example of Test Results iiccsscssosasshooseencteses 14 19 RELELSNCES ssasasanarsiacssaennasscnasaecrsnavarcunandeaunee 18 Intended Use The CycLex Research Product CycLex CaM kinase II Assay Kit is primarily designed to measure the activities of purified Ca Calmodulin dep ndemt protein kinase II CaM kinase II for the rapid and sensitive evaluation of inhibitors or activators The phospho serine monoclonal antibody used in this assay kit has been demonstrated to recognize the phospho serine residue in Syntide 2 which is efficiently phosphorylated by CaM kinase II Additionally column fractions of cultured primar
10. ate Reagent immediately after wash Reagent Stability All of the reagents included in the CycLex Research Product CaM kinase II Assay Inhibitor Screening kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the ATP must be stored at 20 C Coated assay plates should besstored in the original foil bag sealed by the zip lock and containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Cat CY 1173 11 Version 150310 aR CaM kinase II Assay Kit ory cA ycLex 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 CaM kinase II The following protocols have been shown to work with rat cerebellum and enzyme sources and are provided as examples of suitable methods Concentrated or highly purified CaM kinase II should be diluted Tt is strongly advised that the user always perform an initial experiment to determine the proper ilution 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 pr paration should be performed at
11. but not Provided Cat CY E1173 See page 5 section Materials Required but not Provided 1 Following the above table add the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of Diluted CycLex CaM kinase II Positive Controlzto each well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 302C for 30 minutes 2 Follow the Standard Assay steps 5 10 page8 Special considerations when measuring precise CaM activity In order to measure the activity of CaM kinase II correctly Mit is necessary to conduct the control experiment of Inhibitor control at least once for every experiment and Ca CaM 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 samplez when CaM kinase II enzyme activity is in the sample the high level of A450 is not observed in Inhibitor Control ATP minus control and No enzyme control Test Inhibitor aon Positive NY Assay reagents minus enzyme Sample control control control control Kinase Reaction Buffer Ca CaM plus 80 uL 80 uL 80 uL 80 uL Kinase Reaction Buffer Ca CaM minus 80 uL 10X Staurosporine 1 uM 10 pL Buffer 10 uL H20 10 uL 10 uL 10 uL 10 uL Your enzyme fraction 10 uL 10 uL 10 uL H O CycLex CaM kinase II Positive R
12. containing 15 mUnits 10 uL CaM kinase II Positive Control Cat CY E1173 should be included in each assay a 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 1 minutes but should be individually determined for each system After the final addition incubate at 30 C for 15 minutes 5 Stop the reaction by flicking out the contents Alternatively the reactionanay be terminated by the addition of 150 uL 0 1 M Na EDTA pH 8 0 to each well 6 Wash wells five times with Wash Buffer making sure each wells filled completely Remove residual Wash Buffer by gentle tapping or aspiration 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 60 minutes Discard any unused conjugate after use oo Wash wells five times with Wash Buffer makin sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 9 Add 100 uL of Substrate Reagent to each Avell 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 450 550 or 450 595 nm can also be used Read th
13. d All samples should b assayed in duplicate w To assay individual column fractions add 10 uL of each fraction to the wells of the assaygplate on ice Duplicate wells containing 15 mUnits 10 uL CaM kinase II Positive Control Cat CY E1173 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 Ca CaMpplus or Kinase Reaction Buffer Ca CaM minus 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 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 60 minutes Discard any unused conjugate 7 Wash wells five times with Wash Buffer making sur each Well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 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 th same order as the previously added Substrate Reagent 10 Measure absorbance in each well using a spectrophotometric plate reader at 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 u
14. e 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 estimatejthe inhibitory effect on CaM kinase II activity in the test chemicals correctly it is necessary to conduet the control experiment of Solvent control at least once for every experiment and Inhibitomcontrol 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 CaM kinase II 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 3 Cat CY 1173 9 Version 150310 AA YCLEX CaM kinase II Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures Assar reavents Test Solvent Inhibitor y reag sample control control Kinase Reaction Buffer Ca CaM plus 80 uL 80 uL 80 uL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10 uL 10X Staurosporine 1 uM 10 uL CycLex CaM kinase II Positive Control 1 5 m unit uL or your enzyme fraction wpe pd aa TOnL Kinase Reaction Buffer Ca CaM plus See page 7 section Preparation of Working Solution 10X Staurosporine 1 uM See page 5 section Materials Required
15. ed e CaM kinase II Positive Control Available from CycLex Cat CY E1173 The positive co should be added to the first well at 15 m units well Unused CaM kinase II enzyme should be st aliquots at below 70 C 100X Calmodulin 100X Calmodulin is included in CycLex CaM kinase II Positive Co at CY E1173 One vial contains 200 uL of 25 ug mL calmodulin derived from bovine bra 10X Staurosporine 1 uM Staurosporine is available from Sigma Cat S 4400 100 uM stock solution DMSO diluted 1 100 in Kinase Buffer e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with Precision repeating pipettor e Wash bottle or multichannel dispenser for plate washing QO e Microcentrifuge and tubes for sample preparation e Vortex mixer e Plate reader capable of measuring absorbance in 96 well sat dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also ed The plate can also be read at a single wavelength of 450 nm which will give a s a r reading 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality Qy C CY 1173 5 Version 150310 aR CaM kinase II Assay Kit ory aa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Store the CaM kinase II Positive Control at below 70 C and the ATP at 20 C in aliquots Storegall other components at 4 C D
16. l calmodulin 3 9 3 0 0 2 4 6 8 10 EGTA mM gt Fig 6 Effect of broad spectrum kinase inhibitor O ivity of recombinant CaM kinase II 120 110 100 90 Relative Activity control o1 on Q Staurosporine nM C CY 1173 16 Version 150310 CaM kinase II Assay Kit wy Ga NycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Fig 7 RESOURCE Q column elution profile of CaM kinase II from rabbit brain extract 4 0 1000 N e CaCl2 Calmodulin 36 F e CaCl2 Calmodulin ane 2 Se 800 2 8 700 a 2 4 6000 5 A 2 0 500 O 1 6 400 1 2 300 0 8 200 0 4 100 0 0 0 C CY 1173 17 Version 150310 aR CaM kinase II Assay Kit ory 5 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Andy HUDMON1 and Howard SCHULMAN Biochem J 364 593 611 2002 2 Van Eldik L and Watterson M Calmodulin and Signal Transduction Academic Press New_York 1998 3 Erondu N E and Kennedy M B Regional distribution of type II Ca2 calmodulin dependent protein kinase in rat brain J Neurosci 5 3270 3277 1985 4 Soderling T R Biochim Biophys Acta 1297 131 138 1996 5 Kolodziej S J Hudmon A Waxham M N and Stoops J K J Biol Chenmg275 04354 14359 2000 6 Kanaseki T Ike
17. o not expose reagents to excessive light Avoid freeze thaw cycles e Allow all the components to come to room temperature before use e 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 e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residue from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits e 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 e Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containing solutions in compliance with local regulations e Avoid contact with Substrate Solution which contains hydrogen peroxide e Avoid contact with Stop Solution which contains Sulfuric Acid e In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention whenNecessary 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 e CAUTION
18. ream signaling pathways Changes in intracellular calcium can display variable responses ranging from highly localized transient elevations within subcellular structures e g a dendritic spine of a neuron to Ca waves that spfeadjthroughout the cell including the nucleus The most ubiquitous calcium sensing protein is Calmodulin CaM which contains four EF hand motifs with high specificity for binding Ca The Ca aM eomplex interacts with and modulates the functionality of a large number of proteins 2 including seV ral Ser Thr protein kinases The CaM kinase II family is encoded by four genes alpha beta gamma and delta that also exhibit alternative splicing The gamma and delta isoforms are expressed in most tissues whereas the alpha and beta isoforms are most prominent in neural tissues and comprise up to 2 yof the total protein in the hippocampus of rodents and up to 1 of the total protein in the forebrain itself 3 The various CaM kinase II subunits are comprised of an N terminal catalytic xegion Ja central regulatory domain containing an autoinhibitory domain AID and Ca CaM binding motif a variable sequence and the C terminal subunit association domain 4 The holoenzyme is an oligomeric protein comprised of twelve 50 60 kDa subunits arranged as two stacked hexamerieiings 5 6 The C terminal association domains form the central core of each ring with the N terminal catalytic domains projecting outward In the ab
19. sed Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquidjat each step is essential to good performance 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 ar obtained when either O D values do not exceed 0 25 units for the blank no enzyme ontrol or 2 5 units for the CaM kinase II positive control Note 3 If the microplatesreader is not capable of reading absorbance greater than the absorbance of the Weel positive control perform a second reading at 405 nm A new O D values measured at 405m jis used to determine CaM kinase II activity of off scale samples The readings at 405 nm sh ul not replace the on scale readings at 450 nm Cat CY 1173 8 Version 150310 CaM kinase II Assay Kit jde Uss Mi GA y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures Kinetic 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 enzyme samples diluted with Kinase Buffer as needed All enzyme samples should be assayed in duplicate w To assay enzyme sample add 10 uL of each enzyme sample or CaM kinase II Positive Contfol Cat CY E1173 to the wells of the assay plate Duplicate wells
20. sence of bound Ca CaM the CaM kinase II is maintained in an inactive conformation because of an interaction of the AID with the catalytic domain of its own Subunit The Ca CaM complex binds to a sequence that partially overlaps the AID presumably causing a conformational change and thereby disrupting interaction of the AID with the catalytie domain and producing kinase activation Interestingly the sensitivity of CaM kinase II to activation by Ca CaM depends on the subunit composition of the holoenzyme 7 Measurement of CaM kinase II activity The protocol generally regarded as most sensitive for the quantitative measurement of CaM kinase II activity involves incubation of the CaMbkinase II sample with substrate either a natural or synthetic polypeptide such as a Syntide 2 in th presence of Mg and P labeled ATP The reaction is terminated by spotting a sample ontoya phosphocellulose P81 filter paper disc followed by washing extensively to remove uninc rporated radiolabel and the incorporated radioactivity on P81 filter is counted While sensitive this method is labor intensive generates hazardous radioactive waste and depends on a radioisotop jof short half life It is particularly unsuitable when kinase assays are only performed on an infrequent Basis The CycLex Research Product CycLex CaM kinase II Assay Kit uses a peroxidase coupled anti sequence specific phosphoserine monoclonal antibody as a reporter molecule in a
21. to phosphorylate the bound substrate in the presence of Mg and ATP The amount of phosphorylated substrate is measured by binding it with a horseradish peroxidase conjugate of MS 6E6 a anti phospho Syntide 2 monoclonal antibody which then catalyzes the conversion of the chromogenic substrate tetra methylbengirdine PMB from a colorless solution to a blue solution or yellow after the addition of stopping x agent The color is quantified by spectrophotometry and reflects the relative amount of CaM kinase Tf activity in the sample For kinetic analysis the sample containing CaM kinase II is added to the vellSyin 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 CaM kinase II Assay Kit is designed to determine non isotopic kinetic analysis of CaM kinase II Careful attention to extraction and purification methods and the assay protocol will provide the investigator with a reliable tool for the evaluation of CaM kinase II activity Summary of Procedure Add 100 uL of assay mixtute to the wells 4 Incubate forg0 min at 30 C Wash the wells t Add 100 uL of ARP conjugated anti phospho Syntide 2 antibody Incubate for 60 min at room temp Wash thejwells 4 Add 100 UL of Substrate Reagent t Add 100 uL of Stop Solution t Measure absorbance at 450 nm
22. uchi Y Sugiura H and Yamauchi T J Cell Biol 145 1049 1060 1991 7 Brocke L Chiang L W Wagner P D and Schulman H J BiolGhem 274 22713 22722 1999 Related Products CaM kinase II Positive Control Cat CY E1173 Anti Phospho Syntide 2 Monoclonal Antibody Clone MS 6E6 Cat CY M1023 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex cogip URL http wwynacy lex oajp 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 1173 18 Version 150310
23. y cell cell line or tissue homogenate can be assayed for CaM kinase II activity with the CycLex Research Product CycLex CaM kinase II Assay Kit if the appropriate dose of CaM kinase II specific inhibitor is used Applications of this kit include 1 Monitoring the purificafiomof CaM kinase II 2 Screening inhibitors omactivators of CaM kinase II 3 Detecting the effects of pharmacological agents on CaM kinase II activity This assay kit is fon research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt tete all components at 4 C e Don texposexeagents to excessive light Cat CY 1173 1 Version 150310 CaM kinase II Assay Kit jde Urs i GA y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures Introduction Ca CaM dependent protein kinase II CaM kinase II is a ubiquitously expressed protein kinase that transduces elevated Ca signals in cells to a number of target proteins ranging from ion channels to transcriptional activators CaM kinase II has a unique holoenzyme structure and autoregulatory properties that allow it to give a prolonged response to transient Ca signals and to sense ettulax Ca oscillations 1 In neurons CaM kinase II is highly expressed and localized with certain subcellular structures Upon activation it can translocate to excitatory synapses where it regulates number of proteins involved in synaptic transmission and its downst
24. ys 1 assay Kinase Buffer provided 9 2 mL 920 uL 92 uL 20X ATP Solution 0 5 mL 50 pL 5 uL 50X CaCh provided 0 29mi 20 pL 2 uL 100X Calmodulin 0 1 mL 10 pL 1 pL Total 10 mL 1000 uL 100 pL 100X Calmodulin is included in CycL xCaM kinase II positive control Cat CY E1173 a final concentration of Calmodulin shouldibe c a 200 ng mL You will need 80 uL of Kinase Reaction Buffer Ca CaM plus per assay well Mix well Discard any unused Kinase Reaction Buffer Ca CaM plus after use 4 Prepare Kinase Reaction Buffer Ca CaM minus by mixing following reagents 96 assays 10 assays 1 assay Kinase Buffer provided 9 2 mL 920 uL 92 uL 20X ATP Solution 0 5 mL 50 pL 5 uL 50X EGTA provided 0 2 mL 20 uL 2 uL B20 0 1 mL 10 uL 1uL Total 10 mL 1000 uL 100 uL In the case of assaying individual column fractions we recommend you to measure the kinase activity in the absence of Calcium Calmodulin as well as in the presence of these in parallel See Example of Test Result Fig 4 p16 Cat CY 1173 7 Version 150310 CaM kinase II Assay Kit jde Uss Mi GA y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures 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 neede

CY-1173

Contents

Download Pdf Manuals

Related Search

Related Contents